BIOLOGY & SCIENCE

Link Servis Mikroskop

2019-05-20T09:35:00.003+07:00

Berikut link servis mikroskop, terima kasih

article : Classification Organism with three domain - six kingdom Kingdom Classification System

2012-03-17T09:15:00.001+07:00

In the post previous articles click here, classification system into the five kingdom system. Now This divides cellular organisms into:

The Kingdoms of Organisms:

This presentation has taken as standard the "three domain - six kingdom" model of organism diversity, based on 16S ribosomal RNA sequence comparisons, of Carl Woese and co-workers. This divides cellular organisms into:

Bacteria (Eubacteria),
Archaea (Archaebacteria) and
Eukarya (Eukaryotes; further divided into Protista, Plantae, Animalia and Fungi).

Reference:
GJ Olsen and CR Woese (1993). FASEB Journal 7: 113-123.

However, Robert H Whittaker and Lyn Margulis have suggested that organisms should be divided into five kingdoms: that is,

Monera or Procaryotae (prokaryotes, including Bacteria and Archaea)
Protista (eukaryotes with single- or colonial cellular organisation, may be photosynthetic)
Fungi (generally multinucleate eukaryotes with walled syncytia)
Animalia (wall-less eukaryote cells organised into complex organisms)
Plantae (generally autotrophic plants, mostly with photosynthetic plastids, simple multicellular to advanced tissue organisation)

This model includes some "plants" as protists, and makes no distinction between Archaea and Bacteria.

Reference:
RH Whittaker and L Margulis (1978). BioSystems 10: 3-18

To complicate matters, James Lake and co-workers have lately proposed a radical re-structuring of the "universal" phylogenetic tree, to split Archaea into Halobacteria, Methanogens and Eocytes. This would mean there are three major groupings of prokaryotes (Eubacteria + Halobacteria, Methanogens and Eocytes), which could all constitute kingdoms on their own, given each is as unrelated to the others as any are to Eukarya.

Reference:
JA Lake (1991). Trends in Biochemical Sciences 16: 46-50.

There is also a 2-empire / 8-kingdom scheme, with Archaeobacteria and Eubacteria as kingdoms in Empire Bacteria, and 6 kingdoms in Empire Eukaryota:

Archezoa (primitive eukaryotes without chloroplasts or mitochondria)
Protozoa (unicellular wall-less eukaryotes with mitochondria)
Chromista (photosynthetic organisms with chloroplasts within the lumen of the rough ER; eg: diatoms, brown algae, cryptomonads, oomycetes)
Plantae (photosynthetic organisms with chloroplasts within cytoplasm; all land plants, many green algae)
Fungi
Animalia

Reference:
Cavalier-Smith T (1993). Microbiological Reviews 57: 953-994

Source article :

http://www.mcb.uct.ac.za

Article : Classification Organism with Five Kingdom System

2012-03-17T09:03:00.002+07:00

Many classification of living things depends on the criteria used. One of the followingclassifications are made based on certain criteria:

Classification Organism with Five Kingdom Classification System

Once upon a time, all living things were lumped together into two kingdoms, namely plants and animals (at least, that's how I learned it). Animals included every living thing that moved, ate, and grew to a certain size and stopped growing. Plants included every living thing that did not move or eat and that continued to grow throughout life. It became very difficult to group some living things into one or the other, so early in the past century the two kingdoms were expanded into five kingdoms: Protista (the single-celled eukaryotes); Fungi (fungus and related organisms); Plantae (the plants); Animalia (the animals); Monera (the prokaryotes). Many biologists now recognize six distinct kingdoms, dividing Monera into the Eubacteria and Archeobacteria.

All I can say is that the sytem holds true for this week, at least. It might even hold up for a century or two. Accepted systems of classification have changed at a far faster pace than the species have taken to evolve, that's for certain.

Kingdoms are divided into categories called phyla, each phylum is divided into classes, each class into orders, each order into families, each family into genera, and each genus into species. A species represents one type of organism, such as dog, tiger shark, Ameoba proteus (the common amoeba), Homo sapiens (us), or Acer palmatum (Japanese maple). Note that species names should be underlined or written in italics.

Classifying larger organisms into kingdoms is usually easy, but in a microenvironment it can be tricky. If you have had a little biology, a good exercise is to describe individual living things, and to try to classify them as to kingdom.

Monera (includes Eubacteria and Archeobacteria)

Individuals are single-celled, may or may not move, have a cell wall, have no chloroplasts or other organelles, and have no nucleus. Monera are usually very tiny, although one type, namely the blue-green bacteria, look like algae. They are filamentous and quite long, green, but have no visible structure inside the cells. No visible feeding mechanism. They absorb nutrients through the cell wall or produce their own by photosynthesis.

Protista

Protists are single-celled and usually move by cilia, flagella, or by amoeboid mechanisms. There is usually no cell wall, although some forms may have a cell wall. They have organelles including a nucleus and may have chloroplasts, so some will be green and others won't be. They are small, although many are big enough to be recognized in a dissecting microscope or even with a magnifying glass. Nutrients are acquired by photosynthesis, ingestion of other organisms, or both.

Fungi

Fungi are multicellular,with a cell wall, organelles including a nucleus, but no chloroplasts. They have no mechanisms for locomotion. Fungi range in size from microscopic to very large ( such as mushrooms). Nutrients are acquired by absorption. For the most part, fungi acquire nutrients from decaying material.

Plantae

Plants are multicellular and most don't move, although gametes of some plants move using cilia or flagella. Organelles including nucleus, chloroplasts are present, and cell walls are present. Nutrients are acquired by photosynthesis (they all require sunlight).

Animalia

Animals are multicellular, and move with the aid of cilia, flagella, or muscular organs based on contractile proteins. They have organelles including a nucleus, but no chloroplasts or cell walls. Animals acquire nutrients by ingestion.

A "mini-key" to the five kingdoms

Suppose you see something in freshwater that certainly appears to be living. How can you begin to determine what it is? Here is a key (not quite perfect) that you might use to help determine the kingdom to which it belongs.

1. Is it green or does it have green parts?
- Yes - go to 2
- No - go to 3
2. Could be a plant or a protist, or blue-green bacteria. Make sure that the green is really part of the organism, though. An animal might have eaten something green, for example.
- Single-celled? go to 6
- Multicellular? Plantae. Look for cell walls, internal structure. In the compound microscope you might be able to see chloroplasts.
3. Could be a moneran (bacteria), protist, fungus, or animal.
- Single-celled - go to 4
- Multicellular (Look for complex or branching structure, appendages) - go to 5
4. Could be a moneran or a protist. Can you see any detail inside the cell?
- Yes - Protista. You should be able to see at least a nucleus and/or contractile vacuole, and a definite shape. Movement should be present, using cilia, flagella, or amoeboid motion. Cilia or flagella may be difficult to see.
- No - Monera. Should be quite small. May be shaped like short dashes (rods), small dots (cocci), or curved or spiral shaped. The largest them that is commonly found in freshwater is called Spirillum volutans. It is spiral shaped, and can be nearly a millimeter long. Except for Spirillum, it is very difficult to see Monerans except in a compound microscope with special lighting.
5. Animalia or Fungi. Is it moving?
- Yes - Animalia. Movement can be by cilia, flagella, or complex, involving parts that contract. Structure should be complex. Feeding activity may be obvious.
- No - Fungus. Should be branched, colorless filaments. May have some kind of fruiting body (mushrooms are a fungus, don't forget). Usually attached to some piece of decaying matter - may form a fuzzy coating on or around an object. In water, some bacterial infections of fish and other animals may be mistaken for a fungus.
6. Most likely Protista. If it consists of long, unbranched greenish filaments with no apparent structure inside, it is blue-green bacteria (sometimes mistakenly called blue-green algae), a Moneran.

Most green protists are flagellates, that is, they move rapidly with a spiralling motion. Unless you get them to stop, you can't really see the flagella. Watch out for colonial protists, though, such as Volvox, which forms a spinning ball of green cells. Don't be fooled into thinking it is a plant.

Remember, the more you observe the organism, the more sure you can be. Many living things have stages that make them resemble members of another kingdom.

Source article :

http://www.ruf.rice.edu

Article : More Know about Ascaris lumbricoides

2009-06-11T17:33:00.000+07:00

HOSPES & DISEASE  NAME

Ascaris  lumbricoides, common saying 'soil transmitted helmint' is  the largest of the intestinal nematodes parasitizing humans. It has worldwide in  distribution and most prevalent through out the tropics,subtropic,and more  prevalent in the countryside than in the city.The disease's name is  ascariasis.

¨MORFOLOGY

Adult: The adults are  cylindrical in shape, creamy-white or pinkish in color. The female averages  20-35cm in length, the largest 49cm. The male is smaller,  averaging 15-31cm in length and distinctly more slender than the female. The  typical curled tail with a pair sickle like copulatory spines. On the tip of the  head there are three lips, arranged as "a Chinese word “ 品They have a complete digestive tract. Reproductive organs  are tubular. male has a single reproductive tubule. The female has two  reproductive tubules and the vulva is ventrally located at the posterior part of  the anterior 1/3 of the body.  The three lips are seen at  the anterior end. The margin of each lip is lined with minute teeth which are  not visible at this magnification.

Egg : There are three type of the eggs.  They are fertilized eggs, unfertilized eggs and decorticated eggs. We usually  describe an egg in 5 aspects: size, color, shape, shell and  content.

Fertilized eggs:  broad oval in shape, brown in color, an average size 60× 45µm. The shell is thicker and consists of ascaroside,  chitinous layer, fertilizing membrane and mammillated albuminous coat stained  brown by bile. The content is a fertilized ovum. There is a new-moon(crescent)  shaped clear space at the each end inside the  shell.

Unfertilized egg: Longer  and slender than a fertilized egg. The chitinous layer and albuminous coat are  thinner than those of the fertilized eggs without ascaroside and fertilizing  membrane. The content is made of many refractable granules various in  size.

Decorticated eggs: Both  fertilized and unfertilized eggs sometimes may lack their outer albuminous coats  and are colorless.

LIFE CYCLE

This worm lives in the lumen of small  intestine, feeding on the intestinal contents, where the fertilized female lays  eggs. An adult female can produce approximately 240,000 eggs per day, which are  passed in feces. When passed, the eggs are unsegmented and require outside  development of about three weeks until a motile embryo is formed within the egg.  

After the ingestion of embryonated eggs in  contaminated food or drink or from contaminated fingers, host digestive juices  acts on the egg shell and liberate the larva into the small intestine. These  larvae penetrate the intestinal mucosa and enter lymphatics and mesenteric  vessels. They are carried by circulation to the liver, right heart and finally  to the lungs where they penetrate the capillaries into the alveoli in which they  molt twice and stay for 10-14days and then they are carried, or migrate, up the  bronchioles, bronchi, and trachea to the epiglottis. When swallowed, the larvae  pass down into the small intestine where they develop into adults. The time from  the ingestion of embryonated eggs to oviposition by the females is about 60-75  days. The adult worms live for about one year. The ascarid life cycle is as the  following  diagram.

Pathogenitas

There are two phase in ascariasis:  
The blood-lung migration phase of  the larvae: During the migration through the lungs, the larvae may  cause a pneumonia. The symptoms of the pneumonia are low fever, cough,  blood-tinged sputum, asthma. Large numbers of worms may give rise to allergic  symptoms. Eosionophilia is generally present. These clinical manifestation is  also called Loeffler’s syndrome. 
The intestinal phase of the adults.  The presence of a few adult worms in the lumen of the small intestine usually  produces no symptoms, but may give rise to vague abdominal pains or intermittent  colic, especially in children. A heavy worm burden can result in malnutrition.  More serious manifestations have been observed. Wandering adults may block the  appendical lumen or the common bile duct and even perforate the intestinal wall.  Thus complications of ascariasis, such as intestinal obstruction, appendicitis,  biliary ascariasis, perforation of the intestine, cholecystitis, pancreatitis  and peritonitis, etc., may occur, in which biliary ascariasis is the most common  complication.

Diagnosis

¨ The symptoms and signs are for reference  only. The confirmative diagnosis depends on the recovery and identification of  the worm or its egg.

1. Ascaris pneumonitis: examination of sputum for  Ascaris larvae is sometimes successful. 
2. Intestinal ascariasis: feces are examined for the  ascaris eggs.

(1) direct fecal film: it is simple and  effective. The eggs are easily found using this way due  to a large number of the female oviposition, approximately 240,000 eggs per worm  per day. So this method is the first  choice.
(2) brine-floatation method:  
(3) recovery of  adult worms: when adults or adolescents are found in feces or vomit and  tissues and organs from the human infected with ascarids , the diagnosis may be  defined. 

articel : Trichuris trichiura

2009-06-11T17:30:00.000+07:00

Morphology

Adult: the worm looks like a buggy whip, the anterior 3/5 is slender and the posterior 2/5 is thick. It is pinkish gray in color. The female worm is 3-5 cm in length and has a long slender esophageal region. The male is smaller than the female and has a curved tail. The reproductive organs of male and female are all double tubule.

Egg: it is barrel or spindle in shape and 50 x 20µm in size. It is brownish and has a translucent polar plug at either ends. The content of the egg is an undeveloped cell

Life cycle

Female releases 1000 to 7000 unembryonated double-plugged eggs each day.Eggs are deposited onto warm, moist shady soil where they embryonate to the L1 stage.

The emrionate egg is the infective stage and is ingested. L1 hatch from the egg in the small intestine and burrow into the mucosa where they are. Adults migrate to the ileocecal and colon regions. Adult worm is inserted into the wall of the intestine penetrating through the mucosa and into the submucosa where blood is ingested.

Pathogenesis:

1.Light infection: Asymptomatic

2.Middle infection: Clinical manifestations are usually abdominal pain, anorexia, diarrhea, constipation .

3. Heavy infection: Bloody diarrhea, emaciation, prolapse of the anus may occur.

Diagnosis:

Discover the eggs in feces by saturated brine flotation method or direct fecal smear.

Treatment and prevention: Same as those of ascariasis

article : Enterobius vermicularis (Pinworm)

2009-06-11T17:29:00.001+07:00

The pinworms are one of the most common intestinal nematodes. The adult worms inhabit the cecum and colon. Right after mating, the male dies. Therefore, the male worms are rarely seen. The female worms migrate out the anus depositing eggs on the perianal skin. Humans get this infection by mouth and by autoinfection.

Morfology

Adults: The adults look like a pin and are white in color. The female worm measures about 8 to 13 mm in size and is fusiform in shape. The male adult is only 2-5mm. The tail of a male is curved. They die right after mating, thus males are rarely seen. The anterior end tapers and is flanked on each side by cuticular extensions called “ cephalic alae”. The esophagus is slender, terminating in a prominent posterior bulb , which is called esophageal bulb. The cephalic alae and esophageal bulb are important in identification of the species.

Egg: 50 to 60m by 25 µm, persimmon seed-like, colorless and transparent, thick and asymmetric shell, content is a larva.Anal smear showing large numbers of Enterobius eggs under the lower power. In the background are also two Ascaris eggs.

Life cycle

site of inhabitation: cecum and colon , infective stage: embryonated egg and infective route: by mouth, without intermediate host and reservoir host, life span of female adults: 1-2 months.

About one-third of pinworm-infected persons are asymptomatic, The adult worms may cause slight irritation of the intestinal mucosa.

Major symptom is anal pruritus, which associates with the nocturnal migration of the gravid females from the anus and deposition of eggs in the perianal folds of the skin. Restlessness, nervousness, and irritability, probably resulting from poor sleep associated with anal pruritus,. In young girls, migration of the worms may produce vaginitis and salpingitis or granuloma of the peritoneal cavity.

Diagnosis

Diagnosis depends on recovery of the characteristic eggs. The eggs and the female adults can be removed from the folds of the skin in the perianal regions by the use of the cellophane tape method. The examination should be made in the morning, before the patient has washed or defecated.

Treatment & Prevention

Since the life span of the pinworm is less than two months, the major problem is reinfection. Albendazole is the drug of choice. Repeated retreatment may be necessary for a radical cure.

Prevention:
1. treat the patients and carriers
2. individual health
3. public health
4. health education and hygienic habits

Epidemiology

Geographical distribution—cosmopolitan in temperate zones with about 30 to 50% of the population infected. It is more common in white than colored people and more prevalent in children than adults. Enterobiasis is most common where people live under crowded conditions such as orphanages, kindergartens, and large families.

article : More Know about Hookworm

2009-06-11T17:22:00.002+07:00

The hookworms cause hookworm disease, which is one of the five major parasitic disease in China(malaria, shistosomiasis, filariasis, kala- azar and hookworm disease). At least two species of hookworms infect man, Necator americanus and Ancylostoma duodenale. They live in small intestine.

Morfology

Adults: They look like an odd piece thread and are about 1cm. They are white or light pinkish when living. ♀is slightly larger than♂.The male’s posterior end is expanded to form a copulatory bursa.

Eggs: 60×40 µm in size, oval in shape, shell is thin and colorless.

Content is 2-8cells.

The Morphological Differences between Two species of Hookworms

____________________________________________________

A. duodenale N. americanus

______________________________________________________

Size larger smaller

______________________________________________________

Shape single curve, looks like C double curves, looks like S

______________________________________________________

Mouth 2 pairs of ventral teeth 1peir of ventral cutting plates

____________________________________________________________

Copulatory circle in shape oval in shape

Bursa (a top view) (a top view)

____________________________________________________________

Copulatory 1pair with separate 1pair of which unite to form

spicule endings a terminal hooklet

_______________________________________________________

caudal spine present no

_______________________________________________________

vulva position post-equatorial pre-equatorial

_______________________________________________________

Lyfe cycle

1. Final host: man

2. Inf. Stage: Larva 3 or filariform larva

3. Inf. Route: by skin

4. Food: blood and tissue fluid

5. Site of inhabitation: small intestine

6. Life span: Ad 15years, Na 3-7years

7. Blood-lung migration:

skin, cavum, right heart, lungs

nPathogenesis & Clinical manifestation

Larval migration

(1) Dermatitis, known as "ground itch" or "stool poison".The larvae penetrating the skin cause allergic reaction, petechiae 0r papule with itching and burning sensation. Scratching leads to secondary infection.

(2) pneumonitis (allergic reaction), Loeffier's syndrome: cough, asthma, low fever, biood-tinged sputum or hemoptysis, chest-pain, inflammation shadows in lungs under X-ray. These manifestations go on about 2 weeks.

Adults in small intestine

(1) Epigastric pain as that of a duodenal ulcer.

(2) A large worm burden results in microcytic hypochromatic anemia (character manifestation). The symptoms are lassitude, edema, palpitation of the heart. In severe case, death may result from cardiac failure or physical exhaustion.

(3) Allotriophagy (orpica) is due to the lack of trace element iron (4) Amenorrhea, sterility, abortionmay take place in women.

(5) Gastrointestinal bleeding

(6) Infantile hookworm disease

Diagnosis

Criterion: 1. hemoglobin is lower than 120g/L in man,
110g/L in woman. 2. find hookworm egg

Method:

1. saturated brine flotation technique

2. direct fecal smear

3. culture of larvae

Treatment

1. Albendazole
2. Mebedazole

Epidemiology

worldwide distribution. 22-26℃ is the optimal temperature for Ancylostoma duodenale development, Ancylostoma duodenale mainly prevalent in north of China. 31-35℃ is suitable for Necator americanus, it is mainly prevalent in south of China

Prevention

Unified measures:
1. sanitary disposal of night soil,
2. individual protection,
3. health education,
4. cultivate hygienic habits,
5. treat the patients and carriers.

Magister Research

2009-06-11T17:12:00.001+07:00

Study of methane production by methanogenic consortium bacteria with a substrate of Jatropha curcas seed pressed cake

Yati Supriatin, I Nyoman P. Aryantha, Siti Khadijah Chaerun

School of Life Sciences and Technology

Institut Teknologi Bandung

ABSTRACT

The study of biogas production with seed pressed cake of Jatropha curcas as a substrate in a 2.5 L reactor scale aims to know the optimum condition to produce biogas (methane). The research was begun by screening the inoculum source from cow dung, dam sediment, and cow rumen liquid. The potency of single and consortium bacteria to produce biogas with the highest methane concentration was evaluated. After the best microbial agents was determined, the next step was the substrate composition using jatropha seed pressed cake with C/N ratio of 12.6 and jatropha seed pressed cake added with tapioca waste with total C/N ratio of 15. Three variations of inoculums concentrations (5%, 10%, and 20%) with four times repetitions were investigated. The system was inoculated with 30 day old inoculum and observed for 70 days by measuring several physical, chemical, and biological parameters in a batch system reactor. The result shows that natural consortium from Jatiluhur dam sediment produces the highest methane gas concentration. Jatropha seed pressed cake with C/N ratio 12.6 and inoculums concentration of 20% gave the best result. Biogas was produced from 14^th day and then increased until 70^th day with total production of 2.822,25 mL (average 40.31 mL per day). GC analysis showed that methane concentration at day 49^th reached 60.57% and then continued to increase until the 70^th day with a concentration of 74.72%. This value is the highest result compared with other achievements reported in literatures. Aerobic bacteria were isolated in the last process and identified as Bacillus brevis, Bacillus laterosporus, Edwardsiella tarda, Bacillus pasteurii, Bacillus larvae, Bacillus pumilus while, anaerobic bacteria were identified as Desulfotomaculum nigrificans, Alcaligenes faecalis, Desulfotomaculum orientis, Bacillus badius, Bacillus alcalophilus, Bacillus firmus. Biogas was produced the best at a range of pH from 6 - 8. This result can be used as a reference in biogas production with seed pressed cake of J. curcas as a substrate in application scale.

Keywords: biogas, methane, methanogenic bacteria, seed pressed cake, Jatropha curcas.

1. Introduction

The fossil fuel consumption in Indonesia increasingly at several years while production was decreased (Hambali et al, 2006). This problem could be solve with search new alternative energy source. Biogas as gas generated from organic digestion in anaerobic conditions by mixed population of microorganisms is one of alternative energy source, that can be produced from various of wastes. Biogas generally composes of methane (55-65 %), carbondioxide (35-45%), nitrogen (0-3%), hydrogen (0-1%), and hydrogen sulfide (0-1%) (Anunputtikul & Rodtong, 2004). Jatropha seed pressed cake as a bio diesel production waste will be abundant in Indonesia for few years later because developing of bio diesel as one of bio fuel (Hambali et al, 2006). The usage of Jatropha seed pressed cake as substrate to produce biogas had been developed in nicaragua since 1997 (Gubitz, 1997), nevertheless biogas system from Jatropha seed pressed cake have not yet many developed in Indonesia. The aim of this study is to get optimum environment condition in biogas production and the result can be used as a reference in biogas production with Jatropha seed pressed cake as a substrate in application scale.

2. Materials & Method

2.1 Preparation of Substrate

Jatropha seed pressed cake as main substrate in this study were collected from PT Rajawali Nusantara Indonesia Jatitujuh Majalengka. Tapioca waste used as additional substrate was collected from tapioca factory in Tasikmalaya. Total solid (TS), total carbon, total nitrogen, ash, and sulfur content of raw material were determined using standart methods (Oei & Reinhard, 1989).

2.2 Preparation of inoculum source

Inoculum source obtained from cow rumen liquid was collected from animal slaughtering house in Ciroyom, cow dung was collected from dairy cattle ranch in Cimenyan, and Jatiluhur dam sediment deepness 28 metres. Isolation of methanogenic bacteria from 3 inoculum sources have be done to get single methanogenic isolate for compare the biogas production ability with natural consortium bacteria. Isolate screen base on gas production ability (Lay,1994 modified) and than confirm test base on morphology test under fluorescent microscope (Mink & Dugan, 1976). Natural consortium bacteria from three sources kept in a closed container respectively with regular adding a small amount of jatropha seed pressed cake for 14 days and screen base on methane gas production that measured by using gas chromatography at the same time, inoculum source with the higher methane gas production would be kept for 30 days before inoculating the biogas production reactor.

2.3 Biogas production from Jatropha seed pressed cake

The biogas production from Jatropha seed pressed cake was perfomed using the simple-single-state reactor with working volume of 2 liters (L) in batch system. Substrate formulation carried out in two variation that were only Jatropha seed pressed cake with C/N ratio 12.6 and Jatropha seed pressed cake added tapioca waste with mixed C/N ratio 15. Substrate concentration was 10 % (w/v) to water (Hernas, 2007), and inoculum concentration variation was 5%, 10%, 20% (v/v). The biogas fermentation was then operated in four times repetitions at ambient temperature for 70 days by measuring several physical, chemical, and biological parameters.

Change of physical parameters involved odor and texture slurry, flame test. Change of chemical parameters involved COD used bi chromate method (Oei & Reinhard,1989), Measurement of pH used pH meter (Uchida KT-1A), total gas volume used water displacement method (Anunputtikul & Rodtong,2004), and biogas composition used gas chromatography (Shimatdzu, GC8A, Japan) equipped with column Porapak- Q and Mulsife 5A, injector temperature and detector temperature 70⁰C, column temperature 40⁰C, detector type Thermal Conductivity Detector, gas Argon as the carrier gas with speed 30 cc/minute. Change of C/N ratio during fermentation measured by using Khjedahl method for nitrogen content analysis whereas carbon total content analysis by counted difference of dry weight & ash weight (Oei & Reinhard, 1989).

Biological parameters involved change of aerobic and anaerobic bacteria population by using FDA method (Green et al., 2005 modified), bacteria cell enumeration used plate count method (Cappucino & Sherman, 2005), Isolation and identification aerobic and anaerobic dominant culture able bacteria that cultured at seventh day and 70th day used pour plate method and used nutrient agar media (Cappucino & Sherman, 2005). Quantitative Analysis of methanogenic bacteria had be done at seventh day, 30th day and 70th day used viable count method (Lay, 1994 modified).Reactor design showed at figure 1

3. Result & Discussion

3.1 Raw material for biogas production

Jatropha seed pressed cake was bio diesel production waste that produced from Jatropha curcas seed. Some physical and chemical compositions of Jatropha seed pressed cake were analyzed (Table 1).

Table 1 physical and chemical compositions of substrate

No	Composition (% dry weight)	Jatropha seed pressed cake	Tapioca waste
1.	moisture	10.94	12.67
2.	carbon	84.20	77.393
3.	Nitrogen	6.68	0.393
4.	S	4.53	0.0047
5.	TS	92	80.09
6.	Ash	6.7	2.7

3.2 Biogas production from Jatropha seed pressed cake

Result of methanogenic bacteria isolation & screening state was obtained only one isolate bacterium, because of only one isolate that produced, so this research not enables use isolation result as inoculum. Then the research was more developed at usage natural consortium as inoculum. The biggest methane gas production in natural consortium bacteria screening process was achieved by natural consortium bacteria that indigenous in Jatiluhur dam sediment. It happened because Microorganism variety was very height in sediment and it be caused of sediment supports aerobic micro environment forming anaerobic condition (Maier et al., 2003). Methane gas that produced from sediment could be more than cow rumen liquid. According to Madigan et al. (2003) cow rumen liquid had more CO₂ composition that was 65% than CH₄ that only reach 35%, this condition happen because acetotroph methanogenic bacteria in rumen slow growth whereas retention time for the happening of acetate conversion become CH₄ is short time & sintrophyc bacteria fatty acid degrader not play main role in rumen because fatty acid will be adsorb by intestinal wall and enter to circulation system (Madigan et al., 2003).The result of treatment state was shown in figure 2 &3.

the treatment which higher CH₄ gas production was achieved by C/N ratio 12.6 and inoculum concentration 20 %, composition of methane gas more stabilizes from 49th day till 70th day that is the average of 60.57 % ± 0.205 s/d 74.72% ± 0.256 with total gas production 2.822,25 mL and the average of production biogas per day 40.31 mL.

In C/N ratio substrate 12.6 the system more balance between non methanogenic bacteria & methanogenic bacteria activity compare in C/N ratio substrate 15. Change of pH during fermentation process was decreased at initial pH 8 became average 4-5, moreover in C/N ratio 15 pH decreased drastically became 2-3, it cause methane formation was obstructed.

When a biogas plant is newly started, the acid formation become active first, reducing the pH, then methanogenic bacteria start using these acid, increasing the pH back to neutral (Fulford, 1988) , but it not happen in C/N ratio 15 because of acid forming to much for methanogenic bacteria could consumption (Burke,2001), finally organic matter in C/N ratio substrate 15 was not convert in CH₄ gas. In this study result that in C/N ratio 12.6 CH₄ gas production at 42^nd day decreased drastically, it caused by accumulating CO₂ gas as result of aerobic bacteria metabolism that achieved 2,4 x 10⁷ CFU/mL sample , but after that CH₄ gas production increased until 70^th day. This result appropriate with result of methanogenic bacteria quantitative analysis that the amount of methanogenic bacteria achieve 5.1 x 10³ cell/mL then continued to increased until 16 x 10³ cell/mL at 70^th day (figure 4).

The result of aerobic & anaerobic bacteria population show that aerobic⁷ CFU/mL sampel while bacteria population achieved 10 anaerobic bacteria population reaches 10⁴ CFU/mL sample. Aerobic bacteria were isolated in the last process and identified as Bacillus brevis, Bacillus laterosporus, Edwardsiella tarda, Bacillus pasteurii, Bacillus larvae, Bacillus pumilus while, anaerobic bacteria were identified as Desulfotomaculum nigrificans, Alcaligenes faecalis, Desulfotomaculum orientis, Bacillus badius, Bacillus alcalophilus, Bacillus firmus.

Conclusion

1. Inoculum that produce highest methane gas production is natural consortium bacteria from Jatiluhur dam sediment with the average of methane gas production that produced is 7.163 % ± 0.689.

2. Substrate that produce highest methane gas is Jatropha seed pressed cake with C/N ratio 12.6 with result 74.72% ± 0.256.

3. Inoculum concentration that produce highest methane gas is inoculum concentration 20%, composition of methane gas more stabilizes from 49th day till 70th day that is the average of 60.57 % ± 0.205 s/d 74.72% ± 0.256 with total gas production 2,822,25 mLs and the average of production biogas per day 40.31 mL

Refferences

Anunputtikul, W., & Rodtong, S. (2004 ), The Joint International conference on “ Sustainable Energy and Environment (SEE)”. Laboratory Scale Experiment for Biogas Production from Cassava Tubers. : Hua Hin, Thailand.

Buchanan, R., & Gibbons, E. (1974), Bergey’s Manual of Determinative Bacteriology, 8^th ed., The Williams & Wilkins Co., Inc., USA.

Burke, D.A. (2001), Dairy Waste Anaerobic Digestion Handbook. Environmental Energy Company, Hill Street Olympia.

Cappuccino, J.G., & Sherman, N. (2005), Microbiology a Laboratory Manual, 7^th Ed, The Benjamin/Cummings Publishing Company, Inc. San Francisco

Dubey, S.K. (2005), Microbial ecology of methane emission in rice agroecosystem, Applied Ecology and Environmental Research, 3(2), 1-27. Dalam situs :http//www.ecology .kee.hu/pdf/0302_001027.pdf.

Edwards,T., & McBride,B,C. (1975), New Method for the Isolation and Identification of Metanogenic Bacteria. Applied Microbiology. American Society for Microbiology. 29 (4). 540-545

Fulford, D. (1988), Running A Biogas Programme: A handbook.ITDG Publishing., London.

Green, V. S., Stott, D.E., & Diack, M. (2005), Assay for fluorescein diaceate hydrolitic activity : optimization for soil sampes, Soil Biol. & Biochem., 38, 693 – 701

Gubitz, M.G., Mittelbach., & Trabi, M. (1997), Biofuel and Industrial Products from Jatropha curcas. Developed from the Symposium Jatropha curcas 97, Managua, Nicaragua.

Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T., & Williams, S.T. (1994), Bergey’s Manual of Determinative Bacteriology, 9^th ed., The Williams & Wilkins Co., Inc., USA.

Hernas,T., Kelompok Riset Biodiesel ITB. (2007), Biogas dari Bungkil Jarak Pagar , inovasi terbaru dari teknik Kimia ITB. ITB News.

Hambali, E., Muzdalifah, S., Sulistyanto, G., & Lesmana, T.(2006), Diversifikasi Produk olahan Jarak Pagar & Kaitannya dengan CSR Perusahaan Swasta di Indonesia. Eka Cipta Foundation, Bogor.

Lay, Bibiana.W. (1994), Analisis Mikroba di Laboratorium. P.T Raja Grafindo Persada, Jakarta.

Mink, W.R., & Dugan, R.P. (1976), Tentative Identification of Metanogenic Bacteria by Fluorescence Microscopy. Applied Microbiology. American Society for Microbiology. 33 (3). 713-717.

Madigan, M.T., Martinko, J.M.& Parker, J. (2003), Biology of Microorganisms, 9^th Ed, Pearson Education, Inc., USA.

Maier, R.M., Pepper, I.L., & Gerba, C.P. (2003), Environmental Microbiology, Academic Press, San Diego.

Oei, B. L., & Reinhard, J. (1989), Metode Analisa pada Fermentasi Anaerob. Pusat Antar Universitas Bioteknologi Institut Teknologi Bandung.

article : Content the MICROBIOLOGY TOPICS

2009-04-30T08:27:00.003+07:00

This relatively new science began with the erroneous belief in spontaneous generation (abiogenesis) and matured into the germ theory on which today’s understanding of infectious disease is based.

MICROBIOLOGY TOPICS

VIRTUAL MICROBIOLOGY CLASSROOM

Click here to visit the online materials used in an actual college-level Microbiology Course. The Virtual Microbiology Classroom provides access to a wide range of educational resources including Power Point Lectures, Study Guides, Review Questions and Practice Test Questions.

HISTORY OF MICROBIOLOGY

From spontaneous generation (abiogenesis) to modern immunology, this link will take you to a series of articles summarizing the key contributors and discoveries.

SPONTANEOUS GENERATION DEBATE

GERM THEORY OF DISEASE

EARLY HISTORY OF IMMUNOLOGY

CLASSIFYING ORGANISMS (SYSTEMATIC & TAXONOMY)

Linnaeus and Binomial Nomenclature

TYPES OF MICROBES

All life is composed of cells, and only two basic models exist; prokaryotes and eukaryotes. All bacteria are prokaryotic cells. Viruses are acellular, or non-living particles that are able to cause infectious disease.

PROKARYOTES (Bacteria & Archaea)

Prokaryotic Microbes
Classification of Bacteria
What Are Normal Flora? 2/10
Probiotic Bacteria that Promote Digestive Health 11/09

EUKARYOTIC MICROBES

Eukaryotic Microbes

VIRUSES

HOW TO USE A MICROSCOPE

SPO provides a number of articles on how to properly use a compound light microscope. The main page provides descriptions and links to help you easily find the specific information that you are looking for.

BACTERIAL IDENTIFICATION

Microbes are tiny and often colorless. That wouldn't be a problem if we didn't so often have to identify them. Without the ability to ID specific microbes, the treatment of infectious disease would not be possible. The following links provide information on several methods used to sleuth out the identity of different bacteria, including differential staining, specialized media and other metabolic tests.

MICROBIAL CONTROL (How to Kill Microbes)

Control of microbial growth (killing microbes) can be carried out number of ways, through use of chemical, physical and chemotherapeutic (antibiotic, antiviral and antifungal) agents.

Here are links to additional pages each covering a specific microbial control method.

Physical Control of Microbes (Heat, UV radiation)
Chemotherapeutic Control of Microbes (Antibiotics and other meds)
Chemical Control of Microbes (Disinfectants, cleaners like bleach and lysol)

INFECTIOUS DISEASE

So small that they can only be seen with a light microscope or electron microscope, humans have battled these tiny pathogens throughout our history. The following links lead to articles that describe and give examples of infectious diseases caused by eukaryotic pathogens, bacteria, viruses, viroids and prions.

Sources

Image

van Leeuwenhoek, Public Domain, Wiki

article : Bacterial Endospore Stain Protocol

2009-04-30T08:19:00.005+07:00

Differential Test to Identify Bacteria Genera Bacillus & Clostridium

Read more: "Bacterial Endospore Stain Protocol: Differential Test to Identify Bacteria Genera Bacillus & Clostridium" - http://bacteriology.suite101.com/article.cfm/bacterial_endospore_stain#ixzz0E7UCb8xK&A

Before discussing the procedue for staining endospores, it is important to understand what these unique structures are and how the power of the endospore was first dicovered.

Heat Resistant Bacteria

John Tyndall was a 17th century Irish-born English-bred physicist who made many contributions to science, one of which was the discovery that some microbes existed in two forms:

heat-stable form (endospore)
heat-sensitive form (vegetative cell)

Tyndall found that it took either prolonged or intermittent heating to destroy the resistant heat-stable form. The outcome of this research was a method of sterilizing liquid by heating it to boiling point on successive days ("Tyndallization").

"Tyndallization" is useful for sterilization of growth media in science classes and other situations where autoclaves (instruments that use both heat and pressure to sterilize) are not available for pressure sterilization.

What Is an Endospore?

Endospores are produced by very few types of bacteria, most notably the genera Clostridium and Bacillus. These protective structures are made through a process known as sporulation in response to extreme environmental conditions, such as high temperatures, desiccation, chemicals, changes in pH and lack of food.

In the dormant, inert endospore state, bacteria do not metabolize or reproduce, but exist in a type of suspended animation, much like the seeds of plants do. When environmental conditions again become favorable, the endospore germinates, returning the bacterium to its normal active and reproducing metabolic state.

Staining Bacterial Endospores

Normal water-based techniques, such as the Gram stain, will not stain these tough, resistant structures. In order to stain endspores, malachite green must be forced into the spore with heat, in much the same way that carbol fuschsin is forced through the waxy mycolic acid layer of Mycobacterium in the Acid-fast Stain.

The protocol for differentially staining endospores and vegetative cells is as follows:

Place a strip of blotting paper over the slide.
Place the covered slide over a screened water bath and then saturate blotting paper with primary stain malachite green.
Allow the slide to sit over the steaming water bath for 5 minutes, reapplying stain if it begins to dry out.
Remove blotting paper and rinse slide with water until water runs clear.
Flood slide with the counterstain safrinin for 20 seconds and then rinse.
View specimin under oil immersion (magnification of 1000xTM) with a light microscope.

After this staining procedure, the endospores will appear green, having retained the primary stain, malachite green. The vegetative cells (bacteria are in the active, metabolizing state) will appear pink, having retained the counterstain, safrinin.

Problems Interpreting Endospore Stain

It should be noted that any debris on the slide can also take up and hold the green stain. Everything that ends up green on the slide is not necessarily an endospore. Endospores are small and typically oval. Large or irregular globs of green on the slide may be artifacts.

Acid-fast cells, such as members of Mycobacterium and Nocardia have waxy molecules in their cell wall that will take up and retain the malachite green stain when subjected to the endospore staining process. The uniformly green appearance of endospore stained Acid-fast cells doesn’t mean that they produce endospores. These are vegetative cells that have taken up color from the heat driving malachite green into their waxy cell wall.

Additional Microbiology Information

For more information on prokaryotes and cell biology, go to the excellent websites of SPO Virtual Microbiology Classroom and Microbe Wiki.

Sources

Bauman, R. (2005) Microbiology.

Park Talaro, K. (2008) Foundations in Microbiology.

The copyright of the article Bacterial Endospore Stain Protocol in Bacteriology is owned by Tami Port. Permission to republish Bacterial Endospore Stain Protocol in print or online must be granted by the author in writing.

article : Genetic Topic

2009-04-30T07:58:00.003+07:00

Genetics is the branch of biology that examines how traits are passed down from one generation to another. This is the study of heredity and particularly the mechanisms of hereditary, the genetic code of DNA.

SPO GENETICS TOPICS

MOLECULAR GENETICS

Look here for information on nucleic acids and the genetic code; how DNA is copied, proteins made, and how mistakes, called mutations, can happen.

MOLECULAR GENETIC TOPICS INCLUDE:

HUMAN HEREDITY & MENDELIAN GENETICS

Article series explaining how the combo of our parents genes work together to make you that unique person you are. Learn about the relationship between alleles, genes, DNA, genotypes, phenotypes and complete dominance.

Genotype, Phenotype & Simple Inheritance
Complete Genetic Dominance (Eye Color & Myopia)
Simple Genetic Inheritance (Dimples, Chin Cleft & Free Earlobes)
Dominant Genetic Traits (Mid-digital Hair, Hand Clasping and Bent Finger)
Genetics of Pigment and Skin Cancer 5/09

GENETIC TERMINOLOGY

Chromatin, sister chromatids, daughter chromosomes, haploid, diploid, homologues ... confused? these articles will help you sort out the language of genetics.

GENETIC TERMINOLOGY TOPICS INCLUDE:

CELL DIVISION: Mitosis & Meiosis

This link takes you to a series of articles about mitosis (somatic cell division) and meiosis, the generation of gametes (sperm and eggs).

These different types of cell division are part of the life cycle of all sexually reproducing organisms. There is also information regarding how some organisms can reproduce asexually, without a mate; their cell division resulting in offspring that are clones.

CELL DIVISION TOPICS INCLUDE:

MICROBIAL GENETICS

The basic genetics of cellular microbes is the same as for all cells. The nucleic acid, DNA, is the genome of any cell, and it must be replicated to make new DNA whenever the cell is preparing to divide. RNA transitions the genetic message into the building of proteins for cell structure and function. However, bacteria have some unique capabilities which increase their genetic variability, and viruses have genomes that can be very different from those of cells.

Bacteria Horizontal Gene Transfer (Transformation, Transduction, Conjugation) 6/09

Viral DNA and RNA Genomes 8/09

Making Sense of the Genetics of RNA Viruses (Difference Between Positive & Negative Viral RNA Strands) 8/09

Virtual Cell Biology Classroom Genetics Resources

This genetic information is used in an actual college-level cell biology classroom. The following links will take you to lectures, review questions & practice tests relating to various genetics subtopics.

Molecular Genetics Lecture

Cell Division: Mitosis Lecture

Cell Division: Meiosis Lecture

Mendelian Human Genetics Lecture

Human Genetics Worksheet

Genetics & Probability Test Questions

Sources

Image

Karyotype Sanger Institute

Stem Cells: Ryddragyn Wiki Public Domain

article : More about Immunology

2009-04-30T07:52:00.004+07:00

HUMAN IMMUNOLOGY: The Body's Defense System

The human body has many tactics that is employs; continuously working to eliminate potential pathogens from its territory.

IMMUNOLOGY TOPICS INCLUDE:

HISTORY

Early History of Immunology: Discoveries of Edward Jenner and Louis Pasteur

Some of the early discoveries that led to today's vaccines happened almost by chance. The astute observations of Jenner and Pasteur were the foundation of immunology.

NONSPECIFIC IMMUNITY

Nonspecific Immunity: Skin

Our body’s first line of defense includes structures, and chemicals, processes that work to prevent pathogens entering the body. Read more about how the skin and mucous membrane help defend your body from invaders.

Leukocytes:

Leukocytes are white blood cells that can be categorized into one of two groups based on the presence or absence of granules within:

Extracellular Killing

There are two cell types that do their killing extracellularly (outside the cell). These include Natural Killer Lymphocytes(NK cells) and Eosinophils.

The Complement System

The complement system consists of specific proteins circulating in blood plasma. Most are inactive until they are cut by an enzyme and activated. The pattern of sequential activation of complements results in a cascade of events that ultimately protect the body

Content

Bauman, R. (2005) Microbiology.

Campbell, N. and Reece, J. (2005) Biology,Seventh Edition.

Park Talaro, K. (2008) Foundations in Microbiology.

Images

Macrophage, National Science Foundation

Neutrophil engulfing anthrax bacteria, Tim Vickers, Wikim

article : More Know about Evolution Topics

2009-04-30T07:47:00.004+07:00

Pre-Darwinian Evolutionary Theory: John Lamarck, George Cuvier, James Hutton and Charles Lyell

You may believe Darwin to be the alpha and omega on evolutionary thought, but the proponents of Larmackianism, Catastrophism and Uniformitarianism paved the way.

Charles Darwin & Evolution: Natural Selection as the Mechanism of Biological Change

Darwin was not the first to understand that organisms evolve or change over time. His crucial contribution was identifying the mechanism of change, natural selection.

Evolution as Theory & Fact: The Certainty of Evolutionary Change & Theories of Its Mechanism

"It is important to understand that the current questions about how life evolves in no way imply any disagreement over the fact of evolution." - Neil A. Campbell. This article explores the misunderstanding of the work theory as it applies to evolution.

What Is Natural Selection?

Science Prof Online has an entire web page of links and articles on natural selection, the driving force of evolutionary change, first proposed by Charles Darwin. Select the link above to learn more!

DNA Gene Mutations & Evolution: Genetic Mistakes, Natural Selection and Biological Change 5/09

A mutation is an alteration of a gene's DNA sequence. Mutations are usually bad news, but those rare changes that benefit an organism are the raw material of evolution.

See the Evolution Online Bookstore for recommended readings and direct links to purchase books on evolution.

Sources

Images

Cell Endomembrane Illustration : Mariana Ruiz

Other image credits to be updated.

article : What Is Cell Biology?

2009-04-30T07:38:00.002+07:00

All forms of life are made from these tiny units ... cells, and there are only two main types; prokaryotes and eukaryotes. Explore the following topics and learn more about cells and the cellular processes that that are the common denominator shared by all living things.

SPO Cell Biology Topics

Prokaryotic and Eukaryotic Cells

Prokaryotes, the simplest form of life, are evolutionarily ancient, and for billions of years the only form of life. Now the domains Archea and Eubacteria represent the extant (currently living) types of prokaryotic cells.

While prokaryotic cells include bacteria and bacteria-like Archea, eukaryotic cells are... everything else. From the cells that make up your body to the tiny blob-like amoeba, most living things that we are familiar with are eukaryotes. Eukaryotic cells are distinguished from prokaryotes by the presence of a nucleus and other membrane-bound organelles. The following articles provide information specific to eukaryotes.

Chemistry of the Cell

Think that chemistry has nothing to do with biology? Think again. In order to understand cells and cellular processes, you must have at least a very basic understanding of inorganic and organic chemistry.Click on this topic link to access the articles that will bring you up to speed.

Cellular Metabolism

We are all familiar with the term 'metabolism', but what does it really mean? The following articles explain how the cells of our bodies (and cells of all living things) turn food energy into ATP energy that can be used to fuel cellular reactions.

Cell Division - Mitosis and Meiosis

Cell division is required for living things to grow, develop and reproduce. Mitosis produces cells that are clones, identical to the parent cell. Meiosis is the form of cell division that produced gametes (sperm and eggs). See the links below for more detailed information on cell division.

Molecular Genetics

Learn about nucleic acids, the organic molecules that contain and transmit the genetic code to build the vast array of proteins that make you truly unique.

Stem Cells

What are stem cells? How are they collected, stored, grown and transplanted?

This link will take you to an article series that should answer some of your questions regarding embryonic and adult stem cells, umbilical cord blood banking and more.

Virtual Cell Biology Classroom

Materials used to supplement an introductory college-level Cell Biology Course. The Virtual Cell Biology Classroom provides access to a wide range of educational resources including Power Point Lectures, Study Guides, Review Questions and Practice Test Questions

Sources

Images

Epithilial Cells: WikiBooks Cell Biology Text

Cell Endomembrane System: Mariana Ruiz

Macrophage: National Science Council

article : Differential staining for Clostridium bacteria

2009-04-30T07:34:00.003+07:00

The final differential stain that we will be learning in lab is called an Endospore stain 7/09. It is a special staining procedure that allows us to stain both vegetative (active) cell and endospores.

What are Endospores?

Two genera of bacteria, Clostridium and Bacillus, produce endospores. Endospores are tough, resistant structures that allow the bacteria to essentially exist in ‘suspended animation.’ Endospores do not metabolize and do not reproduce but merely exist, much like plant seeds, until exposed to environmental conditions suitable for bacterial growth.

Example of Clostridium bacteria with characteristic drumstick-shaped endospore-producing cells. The dark rod-shaped cells are vegetative, actice cells. The clear ovals are endospores, and the objects consisting of both dark rod and clear oval are vegetative cells producing endospores.

Endospore Sporulation

Endospores are made through a process known as sporulation in response to extreme environmental conditions. Extreme environments include high temperatures, drying out (dessication), extremes in pH, bacteriocidal chemicals and lack of food. When environmental conditions are favorable, the endospore germinates. Upon germination, the cell returns to its normal metabolic state, capable of reproduction.

Staining Endospores

Normal staining techniques will not stain the resistant endospores. Here are the steps that are required:

Malachite Green: The stain, malachite green, is forced into the spore with heat much like the carbol fuschsin was forced through the waxy mycolic acid layer of Mycobacterium. After flooding the slide with malachite green, it is suspended over a boiling water bath for 5 minutes and then rinsed with water.
Safrinin: Once the endospore is stained, the counter stain, Safranin, provides color for the vegetative (i.e. metabolically active) cells.

What Vegetative Cells and Endospores Look Like

Below is a photo of Bacillus bacteria that have been stained using the endospore stain. At the end of this differential staining process the vegetative cells (active, metabolizing cells) are pink and the endospores, if present, are green.

ADDITIONAL ARTICLES ABOUT ENDOSPORE STAINING

Follow the links below to articles explaining the Endospore stain procedure and the differences between vegetative cells and endospores.

Bacterial Endospore Stain Protocol: Differential Test to Identify Bacteria Genera Bacillus & Clostridium 7/09

Endospore staining involves application of a series of dyes. Malachite green stains endospores and safrinin dyes vegetative cells pink. Here's endospore stain procedure.

Differential Staining & Bacterial Controls: Positive & Negative Controls: Gram, Acid-fast & Endospore Stains 7/09

Bacterial controls are often used with differential stains as examples of typical positive and negative stain reactions; helpful references when identifying unknowns.

Sources:

Microbiology class laboratory material appearing on this website is adapted from the Applied Microbiology laboratory manual by Cynthia Schauer.

Images:

Heat fixed slide prepared for Gram staining. Positive control is on the left and negative control is on the right, T. Port.

article : In Vitro Antibiotic Susceptibility Testing

2009-03-12T16:30:00.000+07:00

Performed to determine the susceptibility of the organism isolated from the diseased host; Generate antibiogram(s)

Broth Dilution MIC test

Agar Dilution MIC test

Broth/Agar MBC test

Agar Diffusion (Kirby-Bauer Disk) Test: Measure diameter of zone of inhibition; Read as susceptible, intermediate, or resistant

Factors influencing zones of inhibition on agar

Concentration of bacteria spread onto agar plate
Pathogen susceptibility
Antibiotic diffusion effects
Agar depth
Growth rate
Temperature
Nutrient availability
Drug antagonists

Factors influencing diffusion of antibiotic

Concentration of antibiotic: Kirby-Bauer disks, E-tests have standardized concentrations
Molecular weight of antibiotic
Water solubility of antibiotic
pH and ionization
Binding to agar

Modern Commercial Kits (e.g., E-test strips (up to six) on an agar plate and can be read out as MIC)

article : Physiological and Biochemical Mechanisms of Drug Resistance

2009-03-12T16:25:00.000+07:00

Bacteria may Demonstrate any of Five General Mechanisms of Antibiotic Resistance:

Lack of entry; Decreased cell permeability
Greater exit; Active efflux
Enzymatic inactivation of the antibiotic
Altered target; Modification of drug receptor site
Synthesis of resistant metabolic pathway

These Mechanisms can be Grouped into Three Broad Categories:

Permeability Mechanisms

Lack of entry; Decreased cell permeability

Greater exit; Active efflux

Enzymatic Inactivation of the Antibiotic Altered Target or Pathway

Altered target; Modification of drug receptor site

Synthesis of resistant metabolic pathway

article : Antibiotic Resistance

2009-03-12T16:24:00.000+07:00

Bacterial Resistance to Antibiotics is either:

Intrinsic (inherent) or phenotypic
Acquired via acquisition of foreign resistance genes
Acquired via mutational events in the native genome

Intrinsic Resistance: organism is inherently not susceptible to the antibiotic

Phenotypic Resistance (non-genetic) (e.g., non-growing cells; gram-negative, outer cell membrane)

Genotypic Resistance: Exchange of r-Determinants (Genes that confer resistance to specific antimicrobial agents); Transfer and recombination of resistant mutant genes is possible through normal bacterial genetic exchange mechanisms (conjugation; transduction; transformation)

Plasmids: Multidrug (multiple) resistance is possible; Can cross species barrier and closely related strains may acquire r-determinants

Plasmid: covalently closed circular extrachromosomal DNA

Dispensable
May carry genes for drug resistance; metabolic enzymes; virulence factors (e.g., exotoxins)
Restricted or broad host range
Small size (~5 Md) are non-conjugal; Large (20-200 Md) can be conjugal

Plasmid transfer between cells

Bacterial conjugation ("sex"):Replication and transfer of the conjugal plasmid via cell-to-cell contact through an F-pilus encoded by tra (transfer) genes
Transduction: Transferred by phage
Transformation possible

Transposons (Tn) (plasmid or chromosomal): Genes transferable within a replicon via self-excision; Multidrug resistance is possible; Can cross species barrier

Integrons found on transposons or plasmids; Contains the gene and the site for incorporating resistance genes as cassettes allowing expression of the genes; Multidrug resistance is possible

Selective chromosomal mutations (single drug resistance)

article : Mechanism of Action of Beta-Lactam Antibiotics

2009-03-12T16:22:00.000+07:00

Structures of Beta-Lactam Antibiotics:

Beta-lactam Ring Structure
Structure of Penicillins
Structure of Cephalosporins

Bactericidal Against Actively Growing Cells

Drug Covalently Links to Cytoplasmic Membrane Regulatory Enzymes (a.k.a., Penicillin binding proteins (PBP))

PBPs function in cell to catalyze crosslinking of peptidoglycan chains

PBPs are transpeptidases

Beta-Lactam Antibiotics at Concentrations >MIC:

Bind to PBPs

Disrupt synthesis of peptidoglycan

Resultant release of autolysins (autolytic enzymes)

Autolysins enzymatically degrade cell wall forming spheroplast (osmotically-sensitive cell lacking rigidity of cell wall)

Cells lyse; Bacterial cell killed

For E. coli, at Concentrations <MIC:

Septum formation is interrupted

Filamentous, multinucleated cells are observed with cells continuing to divide but septa (new cell walls that separate daughter cells) do not form

article : Basic Mechanisms of Antibiotic Action and Resistance

2009-03-12T16:19:00.000+07:00

Five Basic Mechanisms of Antibiotic Action against Bacterial Cells:

Inhibition of Cell Wall Synthesis (most common mechanism)
Inhibition of Protein Synthesis (Translation) (second largest class)
Alteration of Cell Membranes
Inhibition of Nucleic Acid Synthesis
Antimetabolite Activity

Inhibition of Cell Wall Synthesis

Beta-Lactams ---> Inhibition of peptidoglycan synthesis (bactericidal)

Resistance --->

(1) fails to cross membrane (gram negatives)

(2) fails to bind to altered PBP’s

(3) hydrolysis by beta-lactamases

Vancomycin ---> Disrupts peptidoglycan cross-linkage

Resistance --->

(1) fails to cross gram negative outer membrane (too large)

(2) some intrinsically resistant (pentapeptide terminus)

Bacitracin ---> Disrupts movement of peptidoglycan precursors (topical use)

Resistance ---> fails to penetrate into cell

Antimycobacterial agents ---> Disrupt mycolic acid or arabinoglycan synthesis (bactericidal)

Resistance --->

(1) reduced uptake

(2) alteration of target sites

Inhibition of Protein Synthesis (Translation)

30S Ribosome site

Aminoglycosides ---> Irreversibly bind 30S ribosomal proteins (bactericidal)

Resistance --->

(1) mutation of ribosomal binding site

(2) decreased uptake

(3) enzymatic modification of antibiotic

Tetracyclines ---> Block tRNA binding to 30S ribosome-mRNA complex (b-static)

Resistance --->

(1) decreased penetration

(2) active efflux of antibiotic out of cell

(3) protection of 30S ribosome

50S Ribosome site

Chloramphenicol ---> Binds peptidyl transferase component of 50S ribosome, blocking peptide elongation (bacteriostatic)

Resistance --->

(1) plasmid-encoded chloramphenicol transferase

(2) altered outer membrane (chromosomal mutations)

Macrolides ---> Reversibly bind 50S ribosome, block peptide elongation (b-static)

Resistance --->

(1) methylation of 23S ribosomal RNA subunit

(2) enzymatic cleavage (erythromycin esterase)

(3) active efflux

Clindamycin ---> Binds 50S ribosome, blocks peptide elongation; Inhibits peptidyl transferase by interfering with binding of amino acid-acyl-tRNA complex

Resistance ---> methylation of 23S ribosomal RNA subunit

Alteration of Cell Membranes

Polymyxins (topical) ---> Cationic detergent-like activity (topical use)

Resistance ---> inability to penetrate outer membrane

Bacitracin (topical) ---> Disrupt cytoplasmic membranes

Resistance ---> inability to penetrate outer membrane

Inhibition of Nucleic Acid Synthesis

DNA Effects

Quinolones ---> Inhibit DNA gyrases or topoisomerases required for supercoiling of DNA; bind to alpha subunit

Resistance --->

(1) alteration of alpha subunit of DNA gyrase (chromosomal)

(2) decreased uptake by alteration of porins (chromosomal)

Metronidazole ---> Metabolic cytotoxic byproducts disrupt DNA

Resistance --->

(1) decreased uptake

(2) elimination of toxic compounds before they interact

RNA Effects (Transcription)

Rifampin ---> Binds to DNA-dependent RNA polymerase inhibiting initiation & Rifabutin of RNA synthesis

Resistance --->

(1) altered of beta subunit of RNA polymerase (chromosomal)

(2) intrinsic resistance in gram negatives (decreased uptake)

Bacitracin (topical) ---> Inhibits RNA transcription

Resistance ---> inability to penetrate outer membrane

Antimetabolite Activity

Sulfonamides & Dapsone ---> Compete with p-aminobenzoic acid (PABA) preventing synthesis of folic acid

Resistance ---> permeability barriers (e.g., Pseudomonas)

Trimethoprim ---> Inhibit dihydrofolate reductase preventing synthesis of folic acid

Resistance --->

(1) decreased affinity of dihydrofolate reductase

(2) intrinsic resistance if use exogenous thymidine

Trimethoprim-Sulfamethoxazole synergism

article : Characteristics of an Ideal Chemotherapeutic Drug (There are No Perfect Drugs)

2009-03-12T16:18:00.000+07:00

Selective Toxicity Against Target Pathogen But Not Against Host

Would like LD₅₀to be high and Minimal Inhibitory Concentration (MIC) and/or Minimal Bactericidal Concentration (MBC) to be low

LD₅₀= Lethal Dose 50%; Measure of drug toxicity/lethality against host

MIC; Measure of the concentration of the antibiotic necessary to inhibit growth of the target pathogen

MBC; Measure of the concentration of the antibiotic necessary to kill the target pathogen

Favorable Pharmacokinetics: Survive in high concentration and reach the target site (site of infection)

Pharmacokinetics: Action of drugs in the body over a period of time including:

Absorption
Distribution
Localization in tissues

Biotransformation (biochem. alterations)
Excretion

Would Like the Drug to be:

Bactericidal (Microbicidal): Kills microbes (-cidal = death or killing)

vs.

Bacteriostatic: Stops growth of microorganisms without killing them (-static = stationary; at rest; stasis)

Spectrum of Activity (Broad vs. Narrow) Coordinated with Diagnosis

For example:

a broad-spectrum antibiotic would be indicated against a polymicrobial infection, e.g., an intrabdominal anaerobic infection

a narrow spectrum antibiotic would be ideal for an infection caused by a single pathogen, e.g., a staphylococcal skin infection.

Lack of "Side Effects"

Able to Cross Outer and Cytoplasmic Membranes

No or Low Level of Antibiotic Resistance in Target Pathogen and Lack of Cross-Resistance in Closely Related Strains

article : History of Antibiotics

2009-03-12T16:17:00.000+07:00

~ 1495 Europeans Mercury ------> syphilis (Treponema pallidum)

?? Chinese Moldy soybean curd -----> boils (furuncles, carbuncles) (S. aureus)

?? S. American Indians Chewed bark of cinchona tree ----> malarial fever (Plasmodium spp.); Wore sandals furry with mold -----> foot infections

~ 1630 Europeans Quinine (bark of cinchona) -----> malaria (Plasmodium spp.)

1877 Louis Pasteur Inhibition of some microbes by others; anthrax (Bacillus anthracis)

1908 Gelmo Synthesized sulfanilamide (1^st sulfonamide)

1908-10 Paul Ehrlich Selective stains; Synthesized arsenic compound arsphenamine; (Nobel Prize) ("606", Salvarsan) -----> syphilis (T. pallidum); Coined terms "magic bullet," "chemotherapy," "chemical knife); Further progress delayed by physician hesitancy

1913 Eisenberg Studied bactericidal properties of azo dyes with sulfonamide grouping

1924 Gratia & Dath Systematic search of soil microorganisms; Discovered actinomycetin from Actinomycetes

1928/9 Alexander Fleming Inhibition of S. aureus colonies by mold Penicillium notatum; (Nobel Prize) Discovered "miracle drug," penicillin

1935 G. Domagk Prontosil (only active in vitro) but converted in the body tosulfanilamide moiety (active in vivo) which was secreted in the urine; analog of vitamin para-aminobenzoic acid

1939 G. Domagk Therapeutic value of sulfonamides (sulfur group of compounds); (Nobel Prize) Streptococcus and broad range antimicrobial activities

1939 Rene Dubos Isolated gramicidin and tyrocidin from Bacillus brevis; Active against gram positive bacteria

By 1945 Pharmacologists had 5488 derivatives of sulfanilamide (sulfonamides)

1944/5 Selman Waksman Isolated streptomycin from Streptomyces griseus (soil microbes); (Nobel Prize) Active against some gram negatives and Mycobacterium tuberculosis; Proposed term "antibiotic" = chemical compound made by a microorganism that inhibits or kills other microorganisms at low concentrations

1945 Florey & Chain Reinvestigated, isolated and characterized penicillin; (Nobel Prize) Demonstrated practical clinical value as better means to prevent death from infection of war wounds

Designed & Maintained by David M. Rollins
Copyright © 2000, D.M. Rollins and S.W. Joseph
Revised: August 2000
URL: http://life.umd.edu

article : Definition of an Antibiotic

2009-03-12T16:14:00.000+07:00

Antibiotic (Literal definition):

Against (anti-) life (-biotic); Often detrimental to host

Antibiotic (Old definition):

Chemical substance produced by various species of microorganisms that is capable, in low concentrations, of inhibiting the growth of or killing other microorganisms

Antibiotic (New definition):

Substance produced by a microorganism or a similar product produced wholly (synthetic) or partially (semi-synthetic) by chemical synthesis and in low concentrations inhibits the growth of or kills microorganisms

article : Emerging Infectious Diseases

2009-03-12T16:11:00.000+07:00

NOVA: JOURNEY TO PLANET EARTH Ecosystem Disruption and Emerging Infectious Diseases
DISEASE	PATHOGEN	GEOGRAPHIC LOCATION**	HOST, SOURCE or RESERVOIR	VECTOR	ECOSYSTEM DISRUPTION
Malaria	Plasmodium spp. (protozoa)	Lake Victoria, East Africa --Kenya, Uganda Tanzania (endemic) Nairobi, Kenya (epidemic) Peruvian mountain jungles & Iquitos, Peru (epidemic: increase from 1500 cases/yr to 165,000)	Mosquitos & infected humans; Mosquito larvae in water sources	Anopheles (mosquito)	Endemic in tropical & subtropical rural zones; Rural fishing economy in decline due to raw sewage, agricultural runoff, overfishing Mobilization to urban areas; People bring malarial parasite and mosquitoes; Lack of sanitation & clean water Clear cutting forests & Overcrowding in urban areas with adjacent agriculture provide ideal conditions for breeding mosquitoes
Dengue Fever	Dengue Virus Flavivirus	Iquitos, Peru	Humans, monkeys	Aedes aegypti ('urbanized' mosquito)	Human encroachment Mosquito breeds in stagnant water found in manmade containers
Cholera	Vibrio cholerae (bacteria)	Bay of Bengal Dakka, Bangladesh (endemic) Coastal fishing village to Lima, Peru (1991) to S.America to N.America (epidemic)	Water contaminated with human feces; Zooplankton (copepods) carry the bacteria (symbiosis)	None	Monsoons (floods); Increases in water temperature with plankton blooms; Overcrowding In Peru, El nino of 1990-92 warmed waters; Lack of sanitation and clean drinking water; Subsequent travel, commerce & tourism spread disease
Yellow Fever	Yellow Fever Virus Flavivirus	Peru	Humans, monkeys	Aedes aegypti ('yellow fever' mosquito)	Human encroachment (ecotourism, airtravel, commerce); People move virus and mosquitoes to other parts of the world; Mosquito breeds in stagnant water found in manmade containers
Oyster diseases(e.g., dermo, MSX) Lesions & death in fish; Diarrhea & memory loss in humans	Perkinsus marinus (dermo) Haplosporidium nelsoni (MSX) (both protozoa) Pfiesteria (toxic algae) and terrestial-source fungi??	Chesapeake Bay Crisfield, Maryland	Brackish warm water	None known	Runoff of land-based pollution?? (agricultural wastes, sewage, pesticides, industrial effluents, etc.)
West Nile Fever (Encephalitis)	West Nile Virus Arbovirus	Africa to New York to contiguous United States	Birds (crows, blue jays, raptors, etc.)	Mosquito	Air travel with stowaway virus-carrying insects??

** Location described in television show (not representative of worldwide geographic distribution)

NOTE: The last 30 years has seen an upsurge in waterborne and insectborne diseases. Estimates of 250 million cases of water-related diarrhea annually with 3-5 million deaths per year (20-30,000 per day), the majority of whom are children. Lack of clean drinking water and basic public hygiene and sanitation practices are at the root of the problem.

article : Summary of Host-Parasite Interactions

2009-03-12T16:09:00.000+07:00

I. NATURAL HABITATS FOR MICROBES

Soil; Water; Air

Animals and Animal Products

II. MICROBIAL FLORA OF HUMAN BODY

Skin: Gram-positive bacteria are most common

Respiratory Tract (actually external to body)

Mouth and oropharynx

Saliva

Teeth and gums (gingiva)

Nose and nasopharynx
Microorganisms can be aspirated into the lower respiratory tract

Larynx, trachea and bronchial tubes (bronchi)

Lungs: Superior and inferior lobes (also middle lobe in right lung); Alveoli

Eye (Conjunctivae)
Ear: Inner, middle and exterior; Exterior commonly colonized

Gastrointestinal Tract (actually external to body): Intestinal flora play a significant role in: Digestion; Vitamin production (e.g., vitamin K); Ecological competition (see below) with potentially pathogenic microorganisms

Esophagus: Not typically colonized
Gastric mucosa of stomach: Acid tolerant organisms
Small intestine: Extends from pylorus to ileo-cecal junction (about 20 feet in length); Colonized by mostly anaerobes

Villi (plural of villus) and microvilli are finger-like projections that protrude through the mucous membrane throughout the length of the small intestine and are responsible for absorption

Peyer’s patch: Aggregations of lymphoid tissue concentrated in the ileum

M (microfold) cells: specialized cells in the Peyer’s patches that sample the microenvironment and uptake foreign antigens for processing by underlying macrophages

Duodenum: Upper portion of small intestine (about 10 inches in length) encompassing the superior, descending, transverse and ascending portions, in that order; Hepatic ducts (from liver), pancreatic duct, and cystic duct (from gallbladder) join and enter into the intestine at the descending duodenum

Jejunum (middle portion): Upper two-fifths of remaining length of small intestine

Ileum (lower portion): Remaining three-fifths of length of small intestine

Large intestine: Extends from ileo-cecal valve to anus (about 5 feet in length); >10¹¹ bacteria per gram of feces with anaerobes 1000-fold more common than other microbes

Cecum (a.k.a., Caecum): Large, blind pouch just posterior to the ileo-cecal junction

Colon: Ascending, transverse, descending, and sigmoid portions

Rectum

Anus

Special conditions of intestinal tract

Intestinal tract of newborn

Antibiotic alteration of flora

Genitourinary Tract

External genitalia
Anterior urethra: Normally colonized by avirulent organisms; May be transiently colonized by fecal organisms that can cause disease; Neisseria gonorrhoeae and Chlamydia trachomatis may cause disease or asymptomatically colonize
Urinary bladder: Not normally colonized; May be transiently colonized with urethral organisms
Vagina: Microbial population influenced by hormones
Cervix: Not normally colonized; Neisseria gonorrhoeae and Chlamydia trachomatis are important pathogens

III. NORMALLY STERILE SITES IN THE HUMAN BODY: Colonization of one of these sites generally involves a defect or breach in the natural defenses that creates a portal of entry

Brain; Central nervous system

Blood; Tissues; Organ systems

Sinuses; Inner and Middle Ear

Lower Respiratory Tract: Larynx; Trachea; Bronchioles (bronchi); Lungs; Alveoli

Kidneys; Ureters; Urinary Bladder; Posterior Urethra

Uterus; Endometrium (Inner mucous membrane of uterus ); Fallopian Tubes; Cervix and Endocervix

IV. ECOLOGY DEFINITIONS

Ecological Niche: Unique environmental position occupied by a particular species, perceived in terms of actual physical space occupied and function performed within the community or ecosystem

Flora (Microbiology Definition) = Microbiota: Microorganisms present in or characteristic of a special location (Flora more generically refers to plants; Fauna generically refers to animals)

Normal flora = Indigenous or resident microbiota: Microbial flora typically occupying a particular niche; Organisms tend to segregate given diversity of environmental conditions; Many normal flora perform important functions for the host, including: digestive and nutritional functions and competition with pathogenic microorganisms
Transient flora: Microbial flora only temporarily associated with a particular niche
Endogenous flora: Microbial flora occupying niches that are in or on the body of the host
Exogenous flora: Microbial flora normally existing externally to the body of the host

V. ECOLOGICAL RELATIONSHIPS

Independence: Living free from the influence, guidance, or control by another organism

Benign Relationship (Commensalism): Carrier

Malignant Relationship (Parasitism): Disease

Benign: Referring to a non-life or non-health threating condition; commensalism between host and parasite
Carrier: Symptomless individual who is host to a pathogenic microorganim and has the potential to pass the pathogen to others
Malignant: Disease tending to become progressively worse (Morbidity = illness) and potentially result in death (Mortality = death)

Microbial Interactions: Complex relationships among species; Neutral, Antagonistic, or Synergistic

Host-Parasite Interactions: Commensalism (+/0); Mutualism (+/+); Parasitism (+/-)

Symbiosis: A relationship in which two dissimilar organisms (Symbiotes, Symbionts) live in close association with one another
Commensalism: A relationship between two species in which one is benefited and the other is not affected, neither negatively nor positively
Mutualism: Mutually beneficial relationship between two species
Parasitism: A relationship between two species in which one benefits (Parasite) from the other (Host); Usually involves some detriment to the host

True pathogen (a.k.a., Strict pathogen): Any microorganism capable of causing disease; An infecting agent

Opportunistic pathogen: A usually harmless microorganism that becomes pathogenic under favorable conditions; Often a member of the normal microbial flora

VI. EPIDEMIOLOGY: Study of factors influencing occurrence, transmission, distribution, prevention and control of disease

Epidemic: Occurring suddenly in numbers clearly in access of normal expectancy
Endemic: Present or usually prevalent in a population or geographic area at all times
Pandemic: Widespread epidemic distributed or occurring widely throughout a region, country, continent, or globally

Acquiring Infectious Agents

Portals (Routes) of entry: Ingestion, inhalation, direct penetration
Carrier state: Symptomless individual (host) that is colonized by a pathogenic microorganism and who has the potential to pass the pathogen to others; Carriage may be transient or (semi-) permanent
Nosocomial infections: Infection acquired in a hospital setting that was not present in the host prior to admission, generally occurring within 72 hours of admission
Opportunistic infections: Infection caused by a normally harmless microorganism when certain predisposing conditions (disease or conditions that increase host susceptibility) exist

Transmission of Disease

Portals of entry/exit
Vector: Living carrier, especially the animal that transfers an infectious agent from one host to another; Commonly an Arthropod
Fomite: Inanimate object capable of transmitting microbes from one host to another, e.g., soiled bed linens, diapers, tissues and handkerchiefs, hospital respiratory equipment, etc.

VII. FACTORS CONTROLLING GROWTH OF ORGANISMS

Nutrient Availabilty: The accessibility of a necessary resource, substance or compound providing nourishment to maintain life, i.e. capable of conversion to energy and structural building blocks

Fastidious: an organism that has complex nutritional or cultural requirements, making isolation and culture more difficult

Physico/Environmental Parameters

Water activity/Osmotic pressure:

Water activity (a_w): Represents the available water

Osmotic pressure (p): Expressed in atmospheres; Reflects the concentration of solute in an aqueous solution

Oxygen: Pathogenic microorganisms may have metabolic oxygen requirements that are Obligate or Facultative, Anaerobic or Aerobic, or somewhere in between, Microaerophilic
pH: Power of hydrogen; Measurement of the amount of hydrogen ion in solution; Logarithm of the reciprocal of the hydrogen ion concentration in an aqueous solution used to express its acidity or alkalinity (0-14)
Temperature:

Psycrophile (psychrophilic): Liking cold temperatures; Optimal growth at 15o to 20oC

Mesophile (mesophilic): Liking moderate temperatures; Optimal growth at 20o to 45oC

Thermophile (thermophilic): Liking elevated temperatures; Optimal growth at 50o to 70oC

Competition: Simultaneous demand by two or more organisms or species for a necessary, common resource or physical space (niche) that is in limited or potentially limited supply, resulting in a struggle for survival

Host Immune System: The cells and tissues involved in recognizing and attacking foreign substances in the body

VIII. PATHOGENICITY vs. VIRULENCE

Pathogenicity: Quality of producing or the ability to produce pathologic changes or disease

Virulence: Measurement of the degree of disease producing ability of a microorganism as indicated by the severity of the disease produced; Measure of the dosage (inoculum size) required to caused a specific degree of pathogenicity; One general standard is the LD50

Virulence factors: Microbial characteristics that enhance the ability to cause disease
Dosage (Inoculum size): Number of pathogenic microorganisms entering the host
LD50 = Lethal Dose 50: Number of microorganisms required to cause lethality in 50% of the test host

IX. INFECTION vs. DISEASE

Infection: Colonization and/or invasion and multiplication of pathogenic microrganisms in the host with or without the manifestation of disease

Colonization: Successful occupation of a new habitat by a species not normally found in this niche
Multiplication: Ability of a microorganism to reproduce and increase in numbers during an infection

Disease: Abnormal condition of body function(s) or structure that is considered to be harmful to the affected individual (host); Any deviation from or interruption of the normal structure or function of any part, organ, or system of the body; Dependent upon the dosage and virulence of the organism/agent and varies inversely with the resistance of the host

X. MICROBIAL PATHOGENICITY

Factors that Influence the Degree of Pathogenicity and the Progression of Infection and Disease

Host factors: Age, sex, ethnicity, nutrition (diet), hormonal status; personal hygiene and immune status; Underlying disease or medical condition; Antibiotic or drug usage; Presence of foreign object (e.g., splinter, catheter, sutures, etc.); Innate differences between hosts
Microbial factors: Bacterial virulence factors; Inoculum size (dosage)
External factors (e.g., crowding; seasonal variations; hygiene, sanitation and public health; food processing, storage and preparation; etc.)

Progression of Infection and Disease

Entrance (Portal of entry)
Colonization (Adherence; Adhesion; Attachment)
Multiplication with or without dissemination (spread)
Penetration (Invasion) (Note: Not all pathogens are invasive)
Signs and symptoms of disease (Morbidity; Mortality)
Resolution or chronic state may be established
Elimination and/or exit of pathogen (Carrier state may be established)

Damage to Host

Direct damage

Tissue damage

Cell components and metabolic by-products
Toxins
Enzymes

Organ necrosis: Sum of morphological changes indicative of cell death and caused by the progressive degradative action of cellular components, metabolic by-products, enzymes and/or toxins

Metabolic Effects: Pathogenic organisms can affect any of the body systems with disruptions in metabolic processes

Indirect Damage: Damage to host from excessive or chronic immune response (immunopathogenesis)

XI. VIRULENCE FACTORS

Colonization Factors

Attachment/Adherence: Close association of bacterial cells and host cells generally characterized by receptors and target sites
Surface Receptors/Target Sites: Receptor sites present on both host (Receptor) and bacterial surfaces (Adhesins)

Adhesins: Bind Specific Host Receptors; Often involve fimbriae as structural cell component; Host cell receptors are often sugar moieties; Lectin: Adhesin specific for polysaccharide target receptor (sugar residues)

Fimbriae (plural): Modern term for short, hair-like, protein (pilin) appendages extending outward from the surface of certain bacteria (formerly and a.k.a., pili)
Pili (plural); Pilus (singular): Short, hair-like protein (pilin) appendages extending outward from the surface of certain bacteria; Term more properly applied to those organelles (F-pilus) responsible for bacterial conjugation (transfer of nucleic acids between closely related strains or species = "bacterial sex")

Invasive Factors: Invasins enable a pathogenic microorganism to enter and spread throughout the cells and/or tissues of the host body; Specific recognition of receptor sites on target cells enhances pathogenic advantage

Degradative Enzymes: Class of protein capable of catalytic reactions

Bacterial growth requires food and energy: Growth is achieved by enzymatic catalysis of catabolic (breakdown) reactions of host tissues (resulting in tissue damage) linked to catalysis of anabolic (buildup) reactions in the bacterial cell

Bacterial and host enzymes both play roles in the disease process

Toxigenicity: the ability of a microorganism to cause disease as determined by the toxin it produces which partly determines its virulence

Toxin-like pyrogenic (fever inducing) cell components (e.g., peptidoglycan and peptidoglycan fragments; Teichoic acid or lipoteichoic acid of Gram-positive cell wall)
Endotoxin: Complex bacterial toxin; Lipid A portion of lipopolysaccharide from Gram-negative cell walls; LPS is composed of Lipid A + Core Polysaccharide + O Antigen (a.k.a., O polysaccharide side chain) and is released upon lysis of the cell during infection (FIGURE 3-11); Lipid A component is responsible for endotoxin activity effects on the host; O side chain is the antigenic portion of the LPS molecule

Effects of endotoxin: Binds to specific receptors on macrophages, B lymphocytes (a.k.a., B cells) and other cells stimulating production and secretion of acute phase immunoreactants and lymphokines (e.g., IFN-gamma, IL-1, TNF-alpha, IL-6, histamine, prostaglandins); Stimulates growth of B cells (mitogenic)

Lymphocyte: Agranular leukocyte that is concentrated in lymphoid tissue and is active in immunological responses in the body, including the production of lymphokines (cytokines) and antibodies

Septic shock (sepsis): Endotoxemia; Endotoxin in the blood; Associated with overwhelming infection resulting in vascular system failure with sequestration of large volumes of blood in capillaries and veins; Activation of the complement and kinin systems and the release of histamines, prostaglandins, and other mediators may be involved
Fever (Pyrogenicity): Any elevation of the body temperature above the normal; Functions to speed up immune reactions and to limit/slow bacterial growth and multiplication
Leukopenia and then Leukocytosis: Abnormal reduction in the number (-penia) of leukocytes in the blood, (specifically a count of 5000 or less per cubic millimeter) / Abnormal increase in the number (-cytosis) of leukocytes in the blood, as during hemorrhage, infection, inflammation, or fever (specifically a count of 12,000 or more per cubic millimeter), respectively
Activation of alternate complement pathway: C3a; C5a
Increased vascular permeability (vasodilation); Decreased peripheral circulation; Decreased perfusion (blood flow) of blood to major organs

Petechiae: Round, purple lesions caused by intradermal or submucosal microvascular hemorrhaging; capillary leakage; microhemorrhage
Hypotension: Low blood pressure
Shock

Effects on metabolic and liver functions

Decreased iron
Hypoglycemia: Abnormally low glucose levels

Activation of clotting pathway

Thrombocytopenia: Abnormally low numbers of blood platelet
Thrombosis: formation of blood clot (thrombus) in heart or blood vessel
(DIC) Disseminated intravascular coagulation: Disorder characterized by a reduction in the elements involved in blood coagulation due to their utilization in widespread blood clotting within the vessels; Late stages marked by profuse hemorrhaging

Shock: Characterized by failure of the circulatory system to maintain adequate blood flow to the vital organs; Symptoms include: Hypotension; Weak pulse; Rapid and shallow breathing; Low body temperature; CNS (central nervous system) effects (e.g., nausea)
Death

Exotoxins: Potent toxic substance formed and secreted extracellularly by species of certain bacteria; Genetic control can be encoded either chromosomally, on a plasmid, or by a lysogenic bacteriophage

Toxoid: Toxin that can be altered with formaldehyde to lose physiological toxicity while retaining antigenicity; used as a vaccine

Bacterial Cytolysins (a.k.a., Cytotoxins; Cytolytic toxins; Cytolytic enzymes): Responsible for hemolysis and tissue necrosis; May be lethal when administered intravenously

Three major types based on mechanism of action:

Hydrolyze membrane phospholipids (e.g., phospho-lipases of Clostridium, Staphylococcus)
Thiol-activated cytolysins (oxygen-labile) alter membrane permeability by binding to cholesterol; e.g., Streptococcus, Clostridium
Detergentlike activity on cell membranes; e.g. Staphylococcus, rapid rate of lysis

Two-Component (Bipartite; Two domain) Toxins (A-B or A-5B): Usually one component is a receptor-binding domain (B) associated with absorption to target cell surface and transfer of active component across cell membrane; Second component is an enzymatic domain (A) (active component) that enzymatically disrupts cell function

Conform to general structural model: Prototype is diphtheria toxin of Corynebacterium diphtheriae

Bipartite structure (B, binding; A, active)
Receptor-mediated endocytosis (host cell uptake and internalization of exotoxin
ADP-ribosylation of intracellular target host molecule (e.g., host EF-2 (elongation factor-2) is ADP-ribosylated by C. diphtheriae exotoxin)

Other types of exotoxins (e.g., PA, EF, LF protein toxins of Bacillus anthracis)

Bacterial Defenses against Host Responses to Infection

Encapsulation and antigenic mimicry, antigenic masking, and antigenic shift are important bacterial defense mechanisms
Evasion or incapacitation of phagocytic and/or immune clearance

Phagocytosis inhibitors: Mechanisms enabling an invading microorganism to resist being engulfed, ingested, and or lysed by phagocytes/ phagolysosomes; Patients with a defective/compromised monocyte-macrophage system (formerly, RES, reticuloendothelial system) are particularly susceptible to infection
Capsule (Slime layer)

Avoid recognition and killing

Inhibit opsonization, chemotaxis, and/or phagocytosis
Inhibit phagolysosomal fusion and/or resist lysosomal killing
Block activation of phagocytes by interferon-gamma

Destroy (lyse) phagocytic cell

Inactivate/evade complement and antibody

Evade alternate complement system
Survive opsonization in presence of complement and PMNs and survive inside phagocytic cells
Avoid antibody or proteolytically cleave immunoglobulins

Avoid immune response by growing intracellularly

Direct invasion of cells
Resist lysosomal enzymes and antibacterial substances and multiply intracellularly
Escape phagosome; Adapt to cytoplasmic growth

Nonspecific T cell activation and/or mast cell stimulation: Bind TCR on T cell and MHCII on APC without presence of antigen; Life-threatening release of excess interleukins and mediators; Toxin-like, autoimmune-like responses or loss of immunoresponsiveness
Induction of excess or chronic inflammation; Fibrosis (walling-off) of site of infection (e.g., granuloma formation seen in mycobacterial infections)
Resistance to antibiotics: Intrinsic resistance; Plasmid-mediated; Chromosomally-mediated

BIOLOGY & SCIENCE

Link Servis Mikroskop

article : Classification Organism with three domain - six kingdom Kingdom Classification System

The Kingdoms of Organisms:

Article : Classification Organism with Five Kingdom System

Monera (includes Eubacteria and Archeobacteria)

Protista

Fungi

Plantae

Animalia

A "mini-key" to the five kingdoms

Article : More Know about Ascaris lumbricoides

articel : Trichuris trichiura

article : Enterobius vermicularis (Pinworm)

article : More Know about Hookworm

Magister Research

article : Content the MICROBIOLOGY TOPICS

article : Bacterial Endospore Stain Protocol

Differential Test to Identify Bacteria Genera Bacillus & Clostridium

Staining Bacterial Endospores

Problems Interpreting Endospore Stain

Additional Microbiology Information

Sources

article : Genetic Topic

article : More about Immunology

article : More Know about Evolution Topics

article : What Is Cell Biology?

article : Differential staining for Clostridium bacteria

article : In Vitro Antibiotic Susceptibility Testing

article : Physiological and Biochemical Mechanisms of Drug Resistance

Bacteria may Demonstrate any of Five General Mechanisms of Antibiotic Resistance:

These Mechanisms can be Grouped into Three Broad Categories:

Lack of entry; Decreased cell permeability

article : Antibiotic Resistance

article : Mechanism of Action of Beta-Lactam Antibiotics

Structures of Beta-Lactam Antibiotics:

article : Basic Mechanisms of Antibiotic Action and Resistance

Five Basic Mechanisms of Antibiotic Action against Bacterial Cells:

Inhibition of Cell Wall Synthesis

Inhibition of Protein Synthesis (Translation)

Alteration of Cell Membranes

Inhibition of Nucleic Acid Synthesis

Antimetabolite Activity

article : Characteristics of an Ideal Chemotherapeutic Drug (There are No Perfect Drugs)

article : History of Antibiotics

article : Definition of an Antibiotic

article : Emerging Infectious Diseases

article : Summary of Host-Parasite Interactions