Patient 2 - Posted by Hui Yan.

Name: Kwan Siew Lan (outpatient)
Age: 28 Gender: Female
Complaints: Diarrhoea
Diagnosis: Enterocolitis
Antibiotic treatment: Nil
Stool specimen is collected from the patient.

Possible Organisms that causes enterocolitis
1. Salmonella sp.
It is a type of enterobacteriaceae. This type of organism causes bloody diarrhea with mucus.
Key Characteristics of Salmonella sp.
- Gram-negative, motile rods
- facultative anaerobes
- Non-lactose fermenters
- Produces H2S
This organism causes Salmonella enterocolitis. Salmonella enterocolitis is an infection in the lining of the small intestine. Salmonella enterocolitis can range from mild to severe diarrheal illness. The infection is acquired through ingestion of contaminated food or water. Any food can become contaminated during preparation if conditions and equipment for food preparation are unsanitary.

2. Shigella sp.
It is a type of enterobacteriaceae. This type of organism causes watery diarrhea in later stage of disease, the stool contains blood, mucus or pus. The most common symptoms are diarrhea, fever, nausea, vomiting, stomach cramps, and straining to have a bowel movement.
Key Characteristics of Shigella sp.
- Gram-negative, non-motile and non-spore forming rods
- Does not produce H2S
- Aerobic microbe
This organism cause Shigellosis. Shigellosis or known as Shigella enterocolitis is a common cause of acute diarrhea in adults.

3. Campylobacter jejuni
It is gram-negative, curved, rod-shaped bacteria. It is motile with a single polar flagellum and it is micro-aerophillic type of microorganism.

Infection with C. jejuni usually results in enteritis (inflammation of small intestines), which is characterised by abdominal pain, diarrhea, fever, and malaise. Diarrhea can vary in severity from loose stools (watery stools) to bloody stools.

4. Entameba histolytica
Entamoeba histolytica is an anaerobic parasitic eukaryote protozoan. It infects predominantly humans and other primates. The active (trophozoite) stage exists only in the host and in fresh feces; cysts survive outside the host in water and soils and on foods, especially under moist conditions on the latter. When swallowed they cause infections by excysting (to the trophozoite stage) in the digestive tract.

5. Clostridial organisms- Clostridium difficile
Clostridial difficile is a species of bacteria of the genus Clostridium which are gram-positive, anaerobic, spore-forming rods. C. difficile is the most significant cause of pseudomembranous colitis, a severe infection of the colon, often after normal gut flora is eradicated by the use of antibiotics.

Since the patient is an outpatient and she does not have any antibiotic treatment, thus Clostridium difficile is not the cause of enterocolitis as it often is caused by antibiotic treatment and commonly, this type of infection is acquired in the hospital.

6. Giardia lamblia
Giardia lamblia (formerly also Lamblia intestinalis and also known as Giardia duodenalis and Giardia intestinalis) is a flagellated protozoan parasite that infects the gastrointestinal tract and causes giardiasis. Infection causes giardiasis, a type of gastroenteritis that manifests itself with severe diarrhea and abdominal cramps. Other symptoms can include bloating, flatulence, fatigue, nausea, vomiting and weight loss. Giardia is a major cause of intestinal disease worldwide.

7. Enteropathogenic E. Coli (EPEC)
EPEC is a gram-negative bacillus (rod-shaped organism). EPEC causes a profuse watery diarrheal disease and it is a leading cause of diarrhea in developing countries for infants.

The patient is 28 years old, thus EPEC is not the cause of enterocolitis.

Thus, possible organisms that causes enterocolitis in Patient 2 are:
- Salmonella sp.
- Shigella sp.
- Campylobacter jejuni
- Entameba Histolytica
- Giardia lamblia

Investigational tests
1. Microscopy test
A. Gram staining
This test is to find out whether the microorganism is gram positive or gram negative and the shape of the organism (coccus or bacillus). After that, relevant biochemical tests can be done to find out the identity of the suspected organisms.

B. Wet mount
This is used for checking for the presence of pus, blood and any parasites in the stool sample and motility of the microbes.

C. Stool ova and cyst
This test is to check for the presence of cyst and/or ova in the stool.

D. Parasite
This test is to check whether there is any presence of parasite in the stool.

2. Culture (Fecal) - allow the microbes to become enriched in numbers – e.g. using peptone and selenite broth.

3. Serology tests such as slide agglutination tests and Widal tests (tube agglutination) – this is to test whether the suspected microorganism reacts to certain antigens such as O, K, H and Vi antigens.

4. Kirby-Bauer test (using antibiotics discs) or known as Antibiotics Susceptibility test – this is to test whether the cause of enterocolitis is caused by the abnormal flora of certain microorganisms that are resistant to antibiotics

5. Other possible tests
A. Using Salmonella-Shigella agar – to find out whether the suspected microorganism is either Salmonella or Shigella sp.

B. Triple-Sugar Iron test – to find out whether the suspected microorganism ferments any of the 3 sugars (lactose, glucose, fructose) and whether it produce gas or not.

C. Using Campylobacter selective media at 42oC, 10% carbon dioxide, 3-4 days incubation – this is to find out whether the Campylobacter sp. is the cause of enterocolitis. This selective media only allows Campylobacter sp. to grow.

D. MacConkey agar – a selective media to grow Salmonella strains.
It is a selective and differential media used to differentiate between Gram negative bacteria while inhibiting the growth of Gram positive bacteria. The addition of bile salts and crystal violet to the agar inhibits the growth of most Gram positive bacteria, making MacConkey agar selective.

E. Xylose lysine deosycholate (XLD)agar – a selective growth media used in the isolation of Salmonella and Shigella species from clinical samples.
XLD also can be used for the culture of stool samples, and contains two indicators. It is formulated to inhibit Gram-positive bacteria, while the growth of Gram-negative bacilli is encouraged. The colonies of lactose fermenters appear yellow.

F. Blood Agar plate (BAP) - Contains mammalian blood (usually sheep), typically at a concentration of 5–10%. BAP are an enriched, differential media used to isolate fastidious organisms and detect hemolytic activity.

G. Hektoen Enteric (HE) - HE agar is designed to isolate and recover fecal bacteria belonging to the Enterobacteriaceae family. HE is particularly useful in isolating Salmonella and Shigella.

Reference
Brooks, G. F., Butel, J. S. & Ornston, L. N.; “Jawetz, Melnick & Adeberg’s Medical Microbiology”, 23rd edition, Appleton & Lange, 2004.

http://www.nlm.nih.gov

Protozoa diseases

In a place so hot and sweaty, the soldiers can easily get infected by these Protozoa;
Plasmodium, Giardia Lamblia, Crytosporidium and Entamoeba Histolytica.

Plasmodium
4 typical plasmodia that infects human includes: P. falciparum, P. vivax, P. malariae, P. ovale

Epidemiology
Generally limited to tropics and subtropics regions.
Relatively uncommon in the temperate zone, although epidemic outbreaks may occur when the largely nonimmune populations of these areas are exposed, usually unstable and relatively easy to control or eradicate.
Tropical malaria is usually more stable, difficult to control, and far harder to eradicate.

Mode of transmission
Transmission to humans occurs through the bite of female anopheles mosquitoes whereby sporozoites in mosquito saliva are injected into humans

Symptoms
-high fever
-chills
-muscle pain
-diarrhea

Clinical Findings
-Fever
Fever occurs and coincides with RBC lysis.
Periodic febrile episodes become obvious, coinciding with lysis of infected RBC.
Periodicity is 48 hours for P. falciparum, P. vivax, & P. ovale infection but 72 hours for P. malariae infection.

-Anemia
Anemia occurs as a result of RBC destruction leading to enlargement of liver and spleen.

-Large scale intravascular hemolysis
This is observed in P. falciparum infection. As a result, massive hemoglobinuria (blackwater fever) is observed due to the release of Hb from the RBC lysed intravascularly. Manifestation of intravascular hemolysis can include acute tubular necrosis and renal failure.

-Cerebral malaria
Cerebral malaria can result if P. falciparum malaria is left untreated.

Diagnosis
-Thick blood film
Microscopic examination of thick blood film stained with Giemsa’s stain at pH 7.2 helps demonstrates the presence of malarial parasites. This preparation concentrates the parasites and permits the detection even of the mild infections.

-Thin blood film
When the thick blood film demonstrates the presence of malarial parasites, further identification and confirmation of the specific malaria parasite could be done via performance of a Giemsa’s stained thin blood film that allows identification of characteristic specific to particular malaria parasites.
Chracteristics
P.falciparum has ring stage trophozoites that are small and is 1/5 of RBC diameter.
It also has 2 chromatin granules and crescentic gametocytes.
P.vivax has large rings that is ½ to 1/3 of RBC diameter and 1 chromatic granule. It has round or oval shape gametocytes.

-Serological methods
Polymerase Chain Reaction to detect Plasmodium nucleic acids or rapid diagnostic test employing the usage of dipsticks with monoclonal antibody specific against the target parasite antigen such as P. falciparum.

Prevention
-Chemoprophylaxis to travelers
-Mosquito net, window screens, protective clothing and insect repellents
-Drainage of stagnant water reduces the breeding areas

Treatment
Antimalarial grugs like Chloroquine. (1, 2)

Giardia Lamblia
Most common cause of waterborne epidemic diarrheal disease.
Common in wilderness areas because many animal carriers shed cysts into water.
Varies in severity

Mode of transmission
Parasitic in the intestines of humans and animals.
2 stages, one of which is a cyst form that can be ingested from contaminated water. Once the cyst enters the stomach, the organism is released into the gastrointestinal tract where it will adhere to the intestinal wall. Eventually the protozoa will move into the large intestine where they encyst again and are excreted in the feces and back into the environment. Once in the body, the Giardia causes giardiasis.

Symptoms
-Diarrhea
-abdominal cramps
-nausea
-weight loss
-general gastrointestinal distress.

Diagnosis
-Antigen testing of the stool. A small sample of stool is tested for the presence of Giardial proteins. The antigen test will identify more than 90% of people infected with Giardia.

-Can be diagnosed by examination of stool under the microscope; however, it takes three samples of stool to diagnose 90% of cases. Despite requiring three samples of stool, microscopical examination of stool identifies other parasites in addition to Giardia that can cause diarrheal illness. Therefore, microscopical examination of stool has value beyond diagnosing giardiasis, for example, it can diagnose other parasites as the cause of a patient’s illness.

-Collection and examination of fluid from the duodenum or biopsy of the small intestine, but these require a good deal of discomfort. The string test is a more comfortable method for obtaining a sample of duodenal fluid. For the string test, a gelatin capsule that contains a loosely-woven string is swallowed. One end of the string protrudes from the capsule and is taped to the patients outer cheek. Over several hours, the gelatin capsule dissolves in the stomach, and the string uncoils, with the last 12 inches or so passing into the duodenum. In the duodenum the string absorbs a small amount of duodenal fluid. The string then is untapped from the cheek and is removed. The collected duodenal fluid is expressed from the string and is examined under the microscope. Although more comfortable than some of the other tests, it is not clear how sensitive the string test is, for example, does it diagnose 60% (not very good) or 90% (very good) of cases of giardiasis.

Prevention
Avoiding contaminated water and the use of slow sand filters in the processing of drinking water

Treatment
Medicinally by quinacrine, metronidazole, and furazolidone. (3, 5, 6)

Cryptosporidium
Mode of transmission
Causes cryptosporidiosis. Spread by the transmission of oocysts via drinking water, which has been contaminated with infected fecal material. Oocysts from humans are infective to humans and many other mammals, and many animals act as reservoirs of oocysts, which can infect humans. Once inside of its host, the oocyst breaks, releasing four movable spores that attach to the walls of the gastrointestinal tract, and eventually form oocysts again that can be excreted.

Symptoms
-diarrhea
-headache
-abdominal cramps
-nausea
-vomiting
-low fever

Diagnosis
Polymerase Chain Reaction

Prevention
-Practice good hygiene
-Avoid water that might be contaminated
-Avoid food that might be contaminated.

Treatment
No treatment against the protozoa, patients will usually recover, but the disease can be fatal in late stage AIDS patients. (4, 5, 6)

Entamoeba histolytica
Mode of transmission
Via contaminated food and water
It is another water-borne pathogen that can cause diarrhea or a more serious invasive liver abscess. Ingested cysts excyst in the intestine and proteolytically destroy the epithelial lining of the large intestine.

Clinical Findings
May be asymptomatic to fulminating dysentery, exhaustive diarrhea, and abscesses of the liver, lungs, and brain.

Treatment
Several antibiotics

Prevention
Avoiding contaminated water, hyperchlorination or iodination can destroy waterborne cysts. (5, 6)

1. Brooks, G.F., Butel, J.S. & Ornston, L.N. (2004). "Jawetz, Melnick & Adeberg's Medical Microbiology", 23rd edition, Appleton & Lange.
2. http://health.yahoo.com/eney/healthwise/hw119119
3. http://www.medicinenet.com/giardia_lamblia/page3.htm
4. http://en.wikipedia.org/wiki/cryptosporidium
5. http://pages.cabrini.edu/sfuller-espie/Microbiology%20Lecture20Outlines/micro_fungal_protozoan_diseases.htm
6. http://udel.edu/

General Description of the Clinical Diagnosis

Basically there are three different types of clinical diagnosis identified among the five patients and they are: Urinary Tract Infection, Enterocolitis and Food Poisoning.

1) Urinary Tract Infection

Urinary tract infection (UTI) is a condition where one or more structures in the urinary tract become infected after bacteria overcome its strong natural defenses.

Among the 3 patients diagnosed with urinary tract infection, 2 were women and 1 was man.

Normally, only the lower part of the urethra is usually colonized by bacteria as the flushing action of the urinary flow protects against ascending infection. As the female urethra is short, urinary tract infection is more common in women.

Epidemiology and Pathogenesis

Dehydration, obstruction, disturbance of the smooth urinary flow or the presence of a foreign body example stone or urinary catheter, may predispose an individual to urinary tract infection. Trauma during sexual intercourse may precipitate infection in women whereas paediatric infection especially in boys are often associated with congenital abnormality e.g. ureteric reflux or urethral valves.

Possible Causes of Urinary Tract Infection

The most commonly isolated pathogens are Escherichia coli and Enterococcus spp.E.coli uses fimbriae to adhere to the urinary epithelium, thereby reducing the risk of being washed away. Infections caused by Proteus spp. Are more likely in patients who have stones as Proteus spp. have urease activity that raises urinary pH, thus encouraging stone formation. Staphylococcus saprophyticus is a common isolate from sexually active females. Many different Gram-negative organisms colonize urinary catheters, often becoming invasive infections.

Clinical Features / Signs and Symptoms

Lower urinary tract infections are characterized initially by urinary frequency, dysuria (dysuria refers to any difficulty in urination and is sometimes accompanied by pain) and suprapubic (above the pubic bone) discomfort, fever may be absent. In pyelonephritis (an ascending UTI that has reached the pyelum (pelvis) of the kidney), fever, loin (part of the body on either side of the backbone, between the ribs and pelvis) pain, renal angle tenderness and signs of septicaemia (sepsis of the bloodstream caused by bacteremia, which is the presence of bacteria in the bloodstream) may be present. In children, elderly and prenatal patients, UTI may be clinically silent. Recurrent infections can result in scarring and renal failure.

2) Enterocolitis

Enterocolitis is the inflammation of the large and small intestines.

Signs and Symptoms

Fever, abdominal swelling, nausea, vomiting and diarrhea.

There are different types of Enterocolitis and they are: salmonella enterocolitis, antibiotic-associated enterocolitis, hemorrhagic enterocolitis, pseudomembraneous enterocolitis, necrotizing enterocolitis (mostly premature babies), neutropenic enterocolitis, etc.

Since the patient is 28 years old, then necrotizing enterocolitis would be the least possible kind.

Antibiotic-associated enterocolitis is developed when treatment with antibiotics alters the bowel flora and results in diarrhea.

Hemorrhagic enterocolitis is an inflammation of the small intestine and colon, characterized by hemorrhagic breakdown of the intestinal mucosa with inflammatory-cell infiltration.

Pseudomembranous enterocolitis is an acute inflammation of the bowel mucosa with the formation of pseudomembranous plaques overlying an area of superficial ulceration, and the passage of the pseudomembranous material in the feces.

Neutropenic enterocolitis: viral diarrhea and yersinia enterocolitis are commonly found in children.

Possible causes of Enterocolitis
- Due to usage of antibiotics
Examples: Chloramphenicol, AK-Chlor, Chloroptic, Ophthochlor, Pentamycetin, Diochloram, Sopamycetin, Cetina, Clorafen, Paraxin, Quemicetina.

- Possible virus or bacteria infection
Examples: Clostridial organisms, fungi organism, campylobacter jejuni, shigella, Enteropathogenic P E.coli.

3) Food poisoning
Food poisoning is the result of eating organisms or toxins in contaminated food.
Food is an important mode of transmission of infectious diarrhea. Bacterial enters the food chain from animal infections, from poor hygiene during butchering, improper cleaning of storage and preparation areas and unclean utensils cause contamination of raw and cooked foods. Hens that are chronically colonized with salmonella produce eggs that may allow the multiplication of bacteria. Transmission of food poisoning is also facilitated where there is poor sanitation. In these situations, infections spread rapidly through the community, causing significant mortality. Cholera is capable of spreading world-wide. The temperature range in which most bacteria grow is between 40 degrees F (5 degrees C) and 140 degrees F (60 degrees C). Undercooking or improper processing of home-canned foods can cause very serious food poisoning.
Signs and Symptoms
Nausea and vomiting, diarrhea, bloody diarrhea, profuse watery diarrhea with consequent risk of dehydration, severe abdominal pain and cramps, fever, neurologic involvement such as paresthesias (a sensation of tingling, pricking, or numbness of a person's skin with no apparent long-term physical effect), motor weakness, visual disturbances, and cranial nerve palsies, autonomic symptoms such as flushing, hypotension, and anaphylaxis (a severe and rapid multi-system allergic reaction), Headache, dizziness, respiratory failure, and urticaria (a relatively common form of allergic reaction that causes raised red skin welts), myalgias (muscle pain), lymphadenopathy (swelling of one or more lymph nodes), appendicitis like presentation, oliguria (decrease production of urine), neck stiffness and meningeal signs.
Possible causes of food poisoning

- Possible bacteria infection
Examples: Staphylococcus aureus, Salmonella, Clostridium perfringens, Clostridium botulinum, Vibrio parahaemolyticus, Bacillus cereus, Listeria, Yersinia enterocolitica, Campylobacter jejuni, Enteropathogenic Escherichia coli.

Below is a link describing the different types of bacteria that might cause food poisoning.
http://aggie-horticulture.tamu.edu

What is the scope of MSc in medical microbiology in India?

M.Sc (master of science) courses in medical subjects including anatomy, physiology, biochemistry, microbiology and pharmacology are offered by Kasturba Medical College (Manipal and Mangalore). These are the only two colleges in Karnataka that offer these courses. Similar courses are offered by Maharashtra, Kerala, Tamilnadu and Kerala universities too. Every year several students from Karnataka and outside take admission in these two colleges. Not many are aware of its value and utility. The fee structure has been on the rise ever since I took my admission.

I shall concentrate only on M.Sc in medical microbiology only. Even though this is a postgraduate course, it is not considered a PG course by (Medical Council of India (MCI). Typically, any PG course lasts only for two years but medical M.Sc courses are offered for three years. The first year is dedicated entirely to learning medical subjects like anatomy, physiology and biochemistry just the way first year MBBS students do. The only difference lies in Anatomy where brain and limbs are not included in the syllabus. After the student clears the first year, he/she can then proceed to the subject of specialty, which lasts for two years.

During the two years of study, the student has to take up 2-3 internal assessment examination and undertake a dissertation. The course, curriculum and system of examination are exactly similar to that of MD a course. The only difference between M.Sc and MD course is the duration, MD student get three years to study the same portion. During the course the student has to present subject and culture seminars besides undertaking a research work leading to submission of dissertation. At the end of two years a final examination would be held, consisting of theory and practical examination. Practical examination is held for three days and the examiner panel consisting of internal as well as external examiners. The pattern of examination is same that of MD examination. Since Manipal University is a deemed one, answer booklets are evaluated in the same university. The successful candidate is then awarded a masters degree in medical microbiology and a certificate is given to this effect at convocation.

The options these students have next is limited. Unreserved candidates securing 60% or above and reserved candidates with 55% can pursue higher education by undertaking doctoral study (PhD). Clearance of UGC CSIR/NET and Gate exams with good results would be added benefit as they would be given preference to those without these exams. Besides, a regular scholarship too would be given. A student can pursue PhD in any institution or university in Karnataka or outside provided it is recognized by MCI. Obtaining a PhD in non-medical universities will not be recognized by MCI. Those not interested in PhD or unable to purse may opt for other studies such as bioinformatics, clinical research, medical transcription, biotechnology, genetics etc. Other less desirable study options includes computer courses. Students not interested in further studies can find employment in diagnostic laboratories, call centers, pharmaceutical companies or biotech companies. If you are lucky you could land a technician job at any hospital in Gulf. Or worse, one can join a medical college as a teacher.

Scope of M.Sc students joining in medical colleges as faculty: In simple words, there is no scope at all. There was a time when there were no takers for paraclinical subjects like microbiology by MBBS graduates, the M.Sc degree holders were in demand. Now almost every college has full admission to MD microbiology. Each year 20-30 MD students pass out of medical colleges from Karnataka alone and the number of M.Sc students from both the colleges too is almost same. With so many MD degree holders around, the scope for M.Sc degree holders is scarce. Many institutions are not appointing M.Sc degree holders these days. Some colleges appoint them as tutors or demonstrators and not even as lecturers. Even if a M.Sc degree holder is appointed as lecturer, he/she is entitled to promotion as Assistant professor after three years, which is wrongly and unjustly denied in several institutions. The pay package in private institutions too may vary with the degree, where M.Sc degree holders may be underpaid. MCI does not recommend promoting M.Sc faculty above the post of Asst. Prof. without a medical PhD. In simple words, a M.Sc faculty can not expect any growth in medical college without a PhD. The post graduate course (M.Sc) is not considered as a post-graduate course by MCI at all. The discrimination between M.Sc and MD degree holders is intense and many times unhealthy. In simple words, M.Sc holders have no place in a medical world. A M.Sc degree holder is branded as "non-medico" and is looked down upon in many cases.

To view the list of differences between MSc & MD microbiology, visit www.microrao.com/msc.htm

It makes sense that a person with no teaching experience has no eligibility to be an examiner. What bewilders me is that while MSc's are considered perfectly capable of teaching undergraduates, they are not considered capable of examining them. What extra skill does one need to be an examiner? Only PhD holders are allowed as examiners. I always wonder how a research study on a narrow topic can confer upon an MSc teacher a "skill" to become an examiner. The reasons for not letting MSc's become examiners are more than what meets the eye. It is simply a denial of opportunity in order to consider themselves (MDs) superior to MSc's.

How does one become bigger than the other? There are two ways to do this; a) outgrow and outperform the others, b) don't let others perform/grow by denying them opportunity. It is obvious that the second one is felt more appropriate. People want "differences" and how do you create differences? By denial of all opportunities and benefits.

Government has a policy of upliftment of backwards by promoting them via reservations. But what is happening in the medical education is quite the opposite. When the need of the hour is to unite and serve the education with a common goal, the system ends up creating differences. The differences are not only highlighted but every attempt is made to keep the difference not only intact but also to widen it. While the motto should have been to "live and let live", the scenario is quite unlike that. Worse, there is no reservation, no rights or no bodies to fight against oppression for MScs.

There is no denial that the two these two groups are not the same. A demand for equality is also uncalled for. Both these come for different backgrounds and different UG degree. Hence, the concept of superiority of MDs over MSc's is bound to occur. There is no point challenging their superiority or demanding equal status. But it is disheartening to see the denial of opportunities just to make these differences obvious. Agreed that the graduate degrees of both these groups are different but the post graduate degrees are qualitatively the same. When the nature of work is based on the PG degree and not UG degree, why should there be discrimination? The discrimination (if any) should be on merits and not based on UG degree. This is exactly similar to the caste based discrimination, race based discrimination that exists in the society. We now have a new “degree based discrimination”, what I call "academic apartheid". When rights are demanded, MSc's are often told bluntly that they already have been given more than what they deserve. Some day someone should thank them for their generosity. When the education sector itself is infested with this kind of academic apartheid, we can't expect much change to occur in the society.

Summary:
Do not pursue M.Sc in any medical subjects and if you must undertake M.Sc course, then; pursue higher studies, do a PhD and never join a medical college as teaching faculty.

Caution: No degree is good or bad. It is up to the individual what one can achieve with it. The purpose of putting up this article is to highlight the ground reality of value medical MSc has in medical institutions. There is indeed better prospects outside it.
Please don't post questions seeking guidance as they will not be entertained anymore.Posted bySridharat1:03 AM

Transformation

involves the uptake of free or naked DNA released by donor by a recipient.
It was the first example of genetic exchange in bacteria to have been discovered. This was first demonstrated in an experiment conducted by Griffith in 1928. The presence of a capsule around some strains of pneumococci gives the colonies a glistening, smooth (S) appearance while pneumococci lacking capsules have produce rough (R) colonies. Strains of pneumococci with a capsule (type I) are virulent and can kill a mouse whereas strains lacking it (type II) are harmless. Griffith found that mice died when they were injected with a mixture of live non capsulated (R, type II) strains and heat killed capsulated (S, type I) strains. Neither of these two when injected alone could kill the mice, only the mixture of two proved fatal. Live S strains with capsule were isolated from the blood of the animal suggesting that some factor from the dead S cells converted the R strains into S type. The factor that transformed the other strain was found to be DNA by Avery, McLeod and McCarty in 1944.

is gene transfer resulting from the uptake by a recipient cell of naked DNA from a donor cell. Certain bacteria (e.g. Bacillus, Haemophilus, Neisseria, Pneumococcus) can take up DNA from the environment and the DNA that is taken up can be incorporated into the recipient's chromosome.

The steps involved in are:
1. A donor bacterium dies and is degraded.
2. A fragment of DNA (usually about 20 genes long) from the dead donor bacterium binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium.
3. Nuclease enzymes then cut the bound DNA into fragments.
4. One strand is destroyed and the other penetrates the recipient bacterium.
3. The Rec A protein promotes genetic exchange (recombination) between a fragment of the donor's DNA and the recipient's DNA.

Some bacteria are able to take up DNA naturally. However, these bacteria only take up DNA a particular time in their growth cycle (log phase) when they produce a specific protein called a competence factor. Uptake of DNA by Gram positive and Gram negative bacteria differs. In Gram positive bacteria the DNA is taken up as a single stranded molecule and the complementary strand is made in the recipient. In contrast, Gram negative bacteria take up double stranded DNA.

Flash animation of conjugation in gram positive bacterium


Significance: occurs in nature and it can lead to increased virulence. In addition is widely used in recombinant DNA technology.

For more information, visit www.microrao.com/micronotes/genetics.pdfPosted bySridharat7:59 PM

Transduction

is the transfer of genetic material among bacteria that is mediated through bacteriophage.

When a lytic bacteriophage infects a susceptible bacterium, it injects its DNA inside the cell. Following this, the phage DNA initiates its own replication begining with DNA replication and followed by sysnthesis of phage capsid components. At the same time, the host (bacterial) chromosome suffers damage and gets broken down to smaller pieces.

Normally, while packaging, phage DNA is incorporated inside phage capsid however on occasion chromosome DNA (roughly of same size)gets incorporated inside the phage. Following lysis of the infected all the phages are released, with few containing bacterial DNA.

When those phages that have chromosomal DNA instead of its own DNA infect another susceptible bacterium, they introduce bacterial DNA.

Such DNA recombines with bacterial chromosome at the region of homology and result in exchange of genes via recombination, thus a property originally possessed by one bacterium is introduced into another bacterium.

See flash video for understanding:

Posted bySridharat9:44 PM

Chemical methods of disinfection

Disinfectants are those chemicals that destroy pathogenic bacteria from inanimate surfaces. Some chemical have very narrow spectrum of activity and some have very wide. Those chemicals that can sterilize are called chemisterilants. Those chemicals that can be safely applied over skin and mucus membranes are called antiseptics.

ALCOHOLS:
Mode of action: Alcohols dehydrate cells, disrupt membranes and cause coagulation of protein.
Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol
Application: A 70% aqueous solution is more effective at killing microbes than absolute alcohols. 70% ethyl alcohol (spirit) is used as antiseptic on skin. Isopropyl alcohol is preferred to ethanol. It can also be used to disinfect surfaces. It is used to disinfect clinical thermometers. Methyl alcohol kills fungal spores, hence is useful in disinfecting inoculation hoods.
Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable

ALDEHYDES:
Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl group, and probably damages nucleic acids. It kills all microorganisms, including spores.
Examples: Formaldehyde, Gluteraldehyde
Application: 40% Formaldehyde (formalin) is used for surface disinfection and fumigation of rooms, chambers, operation theatres, biological safety cabinets, wards, sick rooms etc. Fumigation is achieved by boiling formalin, heating paraformaldehyde or treating formalin with potassium permanganate. It also sterilizes bedding, furniture and books. 10% formalin with 0.5% tetraborate sterilizes clean metal instruments. 2% gluteraldehyde is used to sterilize thermometers, cystoscopes, bronchoscopes, centrifuges, anasethetic equipments etc. An exposure of at least 3 hours at alkaline pH is required for action by gluteraldehyde. 2% formaldehyde at 40oC for 20 minutes is used to disinfect wool and 0.25% at 60oC for six hours to disinfect animal hair and bristles.
Disadvantages: Vapors are irritating (must be neutralized by ammonia), has poor penetration, leaves non-volatile residue, activity is reduced in the presence of protein. Gluteraldehyde requires alkaline pH and only those articles that are wettable can be sterilized.

PHENOL:
Mode of action: Act by disruption of membranes, precipitation of proteins and inactivation of enzymes.
Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified cresol), hexachlorophene, chlorhexidine, chloroxylenol (Dettol)
Applications: Joseph Lister used it to prevent infection of surgical wounds. Phenols are coal-tar derivatives. They act as disinfectants at high concentration and as antiseptics at low concentrations. They are bactericidal, fungicidal, mycobactericidal but are inactive against spores and most viruses. They are not readily inactivated by organic matter. The corrosive phenolics are used for disinfection of ward floors, in discarding jars in laboratories and disinfection of bedpans. Chlorhexidine can be used in an isopropanol solution for skin disinfection, or as an aqueous solution for wound irrigation. It is often used as an antiseptic hand wash. 20% Chlorhexidine gluconate solution is used for pre-operative hand and skin preparation and for general skin disinfection. Chlorhexidine gluconate is also mixed with quaternary ammonium compounds such as cetrimide to get stronger and broader antimicrobial effects (eg. Savlon). Chloroxylenols are less irritant and can be used for topical purposes and are more effective against gram positive bacteria than gram negative bacteria. Hexachlorophene is chlorinated diphenyl and is much less irritant. It has marked effect over gram positive bacteria but poor effect over gram negative bacteria, mycobacteria, fungi and viruses. Triclosan is an organic phenyl ether with good activity against gram positive bacteria and effective to some extent against many gram negative bacteria including Pseudomonas. It also has fair activity on fungi and viruses.
Disadvantages: It is toxic, corrosive and skin irritant. Chlorhexidine is inactivated by anionic soaps. Chloroxylenol is inactivated by hard water.

HALOGENS:
Mode of action: They are oxidizing agents and cause damage by oxidation of essential sulfydryl groups of enzymes. Chlorine reacts with water to form hypochlorous acid, which is microbicidal.
Examples: Chlorine compounds (chlorine, bleach, hypochlorite) and iodine compounds (tincture iodine, iodophores)
Applications: Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic. Iodine can be combined with neutral carrier polymers such as polyvinylpyrrolidone to prepare iodophores such as povidone-iodine. Iodophores permit slow release and reduce the irritation of the antiseptic. For hand washing iodophores are diluted in 50% alcohol. 10% Povidone Iodine is used undiluted in pre and postoperative skin disinfection. Chlorine gas is used to bleach water. Household bleach can be used to disinfect floors. Household bleach used in a stock dilution of 1:10. In higher concentrations chlorine is used to disinfect swimming pools. 0.5% sodium hypochlorite is used in serology and virology. Used at a dilution of 1:10 in econtamination of spillage of infectious material. Mercuric chloride is used as a disinfectant.
Disadvantages: They are rapidly inactivated in the presence of organic matter. Iodine is corrosive and staining. Bleach solution is corrosive and will corrode stainless steel surfaces.

HEAVY METALS
Mode of action: Act by precipitation of proteins and oxidation of sulfydryl groups. They are bacteriostatic.
Examples: Mercuric chloride, silver nitrate, copper sulfate, organic mercury salts (e.g., mercurochrome, merthiolate)
Applications: 1% silver nitrate solution can be applied on eyes as treatment for opthalmia neonatorum (Crede’s method). This procedure is no longer followed. Silver sulphadiazine is used topically to help to prevent colonization and infection of burn tissues. Mercurials are active against viruses at dilution of 1:500 to 1:1000. Merthiolate at a concentration of 1:10000 is used in preservation of serum. Copper salts are used as a fungicide.
Disadvantages: Mercuric chloride is highly toxic, are readily inactivated by organic matter.

SURFACE ACTIVE AGENTS:
Mode of actions: They have the property of concentrating at interfaces between lipid containing membrane of bacterial cell and surrounding aqueous medium. These compounds have long chain hydrocarbons that are fat soluble and charged ions that are water-soluble. Since they contain both of these, they concentrate on the surface of membranes. They disrupt membrane resulting in leakage of cell constituents.
Examples: These are soaps or detergents. Detergents can be anionic or cationic. Detergents containing negatively charged long chain hydrocarbon are called anionic detergents. These include soaps and bile salts. If the fat-soluble part is made to have a positive charge by combining with a quaternary nitrogen atom, it is called cationic detergents. Cationic detergents are known as quaternary ammonium compounds (or quat). Cetrimide and benzalkonium chloride act as cationic detergents.
Application: They are active against vegetative cells, Mycobacteria and enveloped viruses. They are widely used as disinfectants at dilution of 1-2% for domestic use and in hospitals.
Disadvantages: Their activity is reduced by hard water, anionic detergents and organic matter. Pseudomonas can metabolise cetrimide, using them as a carbon, nitrogen and energy source.

DYES
Mode of action: Acridine dyes are bactericidal because of their interaction with bacterial nucleic acids.
Examples: Aniline dyes such as crystal violet, malachite green and brilliant green. Acridine dyes such as acriflavin and aminacrine. Acriflavine is a mixture of proflavine and euflavine. Only euflavine has effective antimicrobial properties. A related dye, ethidium bromide, is also germicidal. It intercalates between base pairs in DNA. They are more effective against gram positive bacteria than gram negative bacteria and are more bacteriostatic in action.
Applications: They may be used topically as antiseptics to treat mild burns.
They are used as paint on the skin to treat bacterial skin infections. The dyes are used as selective agents in certain selective media.

HYDROGEN PEROXIDE (H2O2)
Mode of action: It acts on the microorganisms through its release of nascent oxygen. Hydrogen peroxide produces hydroxyl-free radical that damages proteins and DNA.
Application: It is used at 6% concentration to decontaminate the instruments, equipments such as ventilators. 3% Hydrogen Peroxide Solution is used for skin disinfection and deodorising wounds and ulcers. Strong solutions are sporicidal.
Disadvantages: Decomposes in light, broken down by catalase, proteinaceous organic matter drastically reduces its activity.

ETHYLENE OXIDE
Mode of action: It is an alkylating agent. It acts by alkylating sulfydryl-, amino-, carboxyl- and hydroxyl- groups.
Properties: It is a cyclic molecule, which is a colorless liquid at room temperature. It has a sweet ethereal odor, readily polymerizes and is flammable.
Application: It is a highly effective chemisterilant, capable of killing spores rapidly. Since it is highly flammable, it is usually combined with CO2 (10% CO2

Bacterial spore

In poor growth conditions some bacteria such as Bacillus and Clostridium produce resistant survival forms termed endospores. This process is known as sporulation. s are endospores in contrast to fungal spores, which are usually exospores. Unlike the spores of fungi, s do not serve reproductive function. They are resistant to extreme environmental conditions such as high temperatures, dryness, toxic chemicals (disinfectants, antibiotics), and UV radiation. Once the endospore is formed, the vegetative portion of the bacterium is degraded and the dormant endospore is released. The endospore is able to survive for long periods of time until environmental conditions again become favorable for growth. The endospore then germinates, producing a single vegetative bacterium. Spores can be killed by sterilization methods such as autoclave and hot air oven. Some chemical disinfectants such as formaldehyde and ethylene oxide can also kill spores.

Mechanism of sporulation:
First the DNA replicates and the cell divides asymmetrically. A cytoplasmic membrane septum forms at one end of the cell. A second layer of cytoplasmic membrane then forms around one of the DNA molecules (the one that will become part of the endospore) to form a forespore. Both of these membrane layers then synthesize peptidoglycan in the space between them to form the cortex. Calcium dipocolinate is also incorporated into the forming endospore. A spore coat composed of a keratin-like protein then forms around the cortex. Sometimes an outer membrane composed of lipid and protein and called an exosporium is also formed. Finally, the remainder of the bacterium is degraded and the endospore is released. There is no metabolic activity until the spore is ready to germinate. Single vegetative cell gives rise to a single spore. Sporulation generally takes around 15 hours.

Germination:
Favorable growth conditions signal the process of endospore germination. Germination of a spore results in a break in the spore wall and the outgrowing of a new vegetative cell. The newly formed vegetative cell is capable of growth and reproduction. A single spore upon germination forms a single vegetative cell. Germination occurs in following steps:

Activation: Even in the presence of favorable conditions, the spore will not germinate until its protective spore coat is not damaged. Conditions such as heat, acidity, abrasion or compounds containing free sulphydryl groups activate the spore to germinate.
Initiation: once activated, the spore will germinate provided the environment is suitable. Different signaling effectors exist for different species. Binding of effector stimulates autolytic enzymes that degrade the peptidoglycan of cortex. Water is absorbed and calcium dipicolinate is released.
Outgrowth: once the cortex and outer layers is degraded, a new vegetative cell consisting of spore protoplast and its wall emerges. This is followed by active biosynthetic activity and process terminates with cell division.

The impermeability of the spore coat is thought to be responsible for the endospore's resistance to chemicals. The resistance of endospores is due to a variety of factors:
Calcium-dipicolinate, abundant within the endospore, may stabilize and protect the endospore's DNA. Specialized DNA-binding proteins saturate the endospore's DNA and protect it from heat, drying, chemicals, and radiation. The cortex may osmotically remove water from the interior of the endospore and the dehydration that results is thought to be very important in the endospore's resistance to heat and radiation. DNA repair enzymes contained within the endospore are able to repair damaged DNA during germination.

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www.microrao.com/micronotes/anatomy.pdfPosted bySridharat6:55 PM

Ziehl Neelsen staining

This differential staining method was introduced by Ehrlich in 1882 and was subsequently modified by Ziehl and Neelsen independently. This staining method is useful in staining Mycobacteria in clinical specimens. Mycobacterial cell wall is made up of a waxy material (mycolic acid) that normally does not allow ordinary stains to enter the cell. The staining technique comprises of a primary stain, a decolouriser and a counterstain. The primary stain, which is typically concentrated (strong) carbol fuchsin is made by dissolving the dye basic fuchsin in phenol. Basic fuchsin dissolves better in phenol than in water. Heating the slide softens the waxy material of cell wall and phenolised dye readily enters the cell. Once stained by this method, these bacteria do not readily decolorize by weak mineral acids. Such bacteria are called acid fast bacteria. The non acid fast structures in the smear are then visualized by counterstaining with methylene blue solution. The acid fast bacilli appear pink in colour.

The procedure adopted in our institution is as follows:
The smear is flooded entirely with concentrated carbol fuchsin solution and heated using a spirit lamp from beneath. The heating should be intermittent and should not be intense to boil the solution or dry it completely. Typically, flaming must be stopped once fumes arise and allowed to cool. The solution is then poured off and washed in gentle stream of running tap water. The smear is then covered with few drops of 20% sulfuric acid and allowed to act for 1-2 minutes and then washed in tap water. The process of decolourisation may be repeated until the smear is faintly pink or almost colourless. The smear is then washed in water and counterstained with methylene blue solution and allowed to act for 30 seconds. The slide is then washed in water and dried with blotting paper and observed under oil immersion objective.

A positive sputum sample typically contain pink coloured, rod shaped bacteria that are slightly curved, sometimes branching, sometimes beaded in appearance, present singly or in small clumps against a blue background of pus cells and epithelial cells.

For more information and commonly asked questions, visit www.microrao.com/staining.htm

Photo of acid fast bacilli in sputum smear

Gram Staining

A differential staining technique was introduced by Hans Christian Gram in 1884, which is now known as Gram Stain technique. The technique comprises of a primary stain (typically crystal violet), a mordant (Gram’s Iodine), a decoloriser (ethyl alcohol) and a counterstain (dil. carbol fuschin). This technique exploits fundamental physiological differences between gram positive bacteria and gram negative bacteria. Once stained by primary stained and fixed by a mordant Gram positive bacteria resists decolorisation by alcohol and remain violet at the end of staining. Gram negative bacteria gets decolourised and must be stained by a counterstain and appear pink in colour at the end of staining. Although the original method devised by Christian Gram comprised of Gentian violet, Lugol’s Iodine, absolute alcohol and Bismarck brown, there are various modifications of Gram stain in practice. These modifications include Jensen’s modification, Kopeloff & Beerman’s modification, Weigert’s modification and Preston & Morrell’s modification.

The procedure adopted in our institution is as follows:
The smear is covered with few drops of crystal violet solution and allowed to act for one minute. The slide is then washed with gentle stream of running tap water. The smear is then covered with few drops of Gram’s Iodine and allowed to act for a minute and then washed in tap water. The smear is then decolourised by alcohol by until no more violet colour comes off the slide. This process is completed within 30 seconds to prevent overdecolourisation. The slide is washed in water and counterstained using dilute carbol fuchsin for 30 seconds. The slide is then washed in water and dried with blotting paper and observed under oil immersion objective.

For more information and commonly asked questions, visit www.microrao.com/staining.htm

Photo of Gram stained smear showing gram positive cocci and gram negative bacilli

Gram stain

The ing method is named after the Danish bacteriologist Hans Christian Gram (1853 –1938) who originally devised it in 1882 (but published in 1884), to discriminate between pneumococci and Klebsiella pneumoniae bacteria in lung tissue. It is a differential staining method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. This reaction divides the eubacteria into two fundamental groups according to their stainability and is one of the basic foundations on which bacterial identification is built. ing is not used to classify archaea, since these microorganisms give very variable responses.

ing consists of four components:
Primary stain (Crystal violet, methyl violet or Gentian violet)
Mordant (Gram's Iodine)
Decolourizer (ethyl alcohol, acetone or 1:1 ethanol-acetone mixture)
Counterstain (Dilute carbol fuchsin, safranin or neutral red)

The original description of staining technique by Christian Gram in a publication titled "The differential staining of Schizomycetes in tissue sections and in dried preparations" in Fortschitte der Medicin; 1884, Vol. 2, pages 185-189 was slightly different from what we use today. The primary stain used was aniline gentian violet, mordant was Lugol's iodine (iodine-potassium iodide in water), decolorizer was absolute alcohol and bismark brown was the counterstain.

Procedure:

The smear on a glass slide is covered with few drops of one of the primary stains. Gentian violet is a mixture of methyl violet and crystal violet. The primary stain renders all the bacteria uniformly violet. After a minute of exposure to the staining solution, the slide is washed in water.

The smear is treated with few drop of Gram's Iodine and allowed to act for a minute. This results in formation of a dye-iodine complex in the cytoplasm. Gram's iodine serves as a mordant.

The slide is again washed in water and then decolorized in absolute ethyl alcohol or acetone. A mixture of ecetone-ethyl alcohol (1:1) can also be used for decolorization. The process of decolorization is fairly quick and should not exceed 30 seconds for thin smears. Acetone is a potent decolorizer and when used alone can decolorize the smear in 2-3 seconds. A mixture of ethanol and acetone acts more slowly than pure acetone. Decolorization is the most crucial part of ing and errors can occur here. Prolonged decolorization can lead to over-decolorized smear and a very short decolorization period may lead to under-decolorized smear.

After the smear is decolorized, it is washed in water without any delay. The smear is finally treated with few drops of counterstain such as dilute carbol fuchsin, neutral red or safranin.

The slide is washed in water; excess water is removed using a blotting paper, dried in air and heat fixed before observing under microscope.

Bacterial culture media

Introduction

There are various reasons why bacteria have to be grown (cultured) in the laboratory on artificial culture media. One of the most important reasons being its utility in diagnosing infectious diseases. Isolating a bacterium from sites in body normally known to be sterile is an indication of its role in the disease process. Indeed, isolating an organism from the clinical specimen is the first step in proving its role as an etiologic agent. Culturing bacteria is also the initial step in studying its morphology and its identification. Bacteria have to be cultured in order to obtain antigens from developing serological assays or vaccines. Certain genetic studies and manipulations of the cells also need that bacteria be cultured in vitro. Culturing bacteria also provide a reliable way estimating their numbers (viable count). Culturing on solid media is another convenient way of separating bacteria in mixtures.

Bacteria infecting humans (commensals or pathogens) are chemoorganoheterotrophs. When culturing bacteria, it is very important to provide similar environmental and nutritional conditions that exist in its natural habitat. Hence, an artificial culture medium must provide all the nutritional components that a bacterium gets in its natural habitat. Most often, a culture medium contains water, a source of carbon & energy, source of nitrogen, trace elements and some growth factors. Besides these, optimum pH, oxygen tension and osmolarity too have to be taken into consideration.

Ingredients
Some of the ingredients of culture media include water, agar, peptone, casein hydrolysate, meat extract, yeast extract and malt extract. While tap water is suitable for culture media, it must not be used if it contains high amount of minerals. In such situations, distilled or demineralised water should be used. Peptone is a byproduct of protein (plant or animal) digestion. Proteins are often obtained from heart muscle, casein, fibrin or soya flour and is digested using proteolytic enzymes such as pepsin, trypsin or papain. The final product contains peptones, proteoses and amino acids besides a variety of inorganic salts including phosphates, potassium and magnesium. Casein hydrolysate is obtained from hydrolysis of milk protein casein using HCl or trypsin. Meat extract is obtained by hot water extraction of lean beef and then concentrated by evaporation. Meat extract contains gelatin, albumoses, peptrones, proteoses, amino acids, creatinine, purines, and accessory growth factors. Yeast extract is prepared from washed cells of bakers’ yeast and contains wide range of amino acids, growth factors and inorganic salts. Malt extract is prepared by extracting soluble materials from sprouted barley in water at 55oC and concentrated by evaporation. It contains maltose, starch, dextrin, glucose and small amounts of protein and protein breakdown products and growth factors.

Brief history
Initially, culture media were very simple; Louis Pasteur used simple broths made up of urine or meat extracts. Robert Koch realized the importance of solid media and used potato pieces to grow bacteria. It was on the suggestion of Fannie Eilshemius, wife of Walther Hesse (who was an assistant to Robert Koch) that agar was used to solidify culture media. Before the use of agar, attempts were made to use gelatin as solidifying agent. Gelatin had some inherent problems; it existed as liquid at normal incubating temperatures (35-37oC) and was digested by certain bacteria.

Classification
can be classified in at least three ways; Based on consistency, based on nutritional component and based on its functional use.
Classification based on consistency:
Culture media are liquid, semi-solid or solid. Liquid media are sometimes referred as “broths” (e.g nutrient broth).
Liquid media are available for use in test-tubes, bottles or flasks. In liquid medium, bacteria grow uniformly producing general turbidity. Certain aerobic bacteria and those containing fimbriae (Vibrio & Bacillus) are known to grow as a thin film called ‘surface pellicle’ on the surface of undisturbed broth. Bacillus anthracis is known to produce stalactite growth on ghee containing broth. Sometimes the initial turbidity may be followed by clearing due to autolysis, which is seen in penumococci. Long chains of Streptococci when grown in liquid media tend to entangle and settle to the bottom forming granular deposits but with a clear medium. Culturing bacteria in liquid media has some drawbacks. Properties of bacteria are not visible in liquid media and presence of more than one type of bacteria can not be detected. Liquid media tend to be used when a large number of bacteria have to be grown. Culture media are suitable to grow bacteria when the numbers in the inoculum is suspected to be low. Inoculating in the liquid medium also helps to dilute any inhibitors of bacterial growth. This is the practical approach in blood cultures. Culturing in liquid medium can be used to obtain viable count (dilution methods).

Solid media:
Any liquid medium can be rendered by the addition of certain solidifying agents. Agar agar (simply called agar) is the most commonly used solidifying agent. The word "agar" comes from the Malay word agar agar (meaning jelly). It is also known as kanten, China grass, or Japanese isinglass. Agar is chiefly used as an ingredient in desserts throughout Japan. It is an unbranched polysaccharide obtained from the cell membranes of some species of red algae such as the genera Gelidium and Gracilaria, or seaweed (Sphaerococcus euchema). Commercially it is derived primarily from Gelidium amansii. Agar is composed of two long-chain polysaccharides (70% agarose and 30% agarapectin). It melts at 95oC (sol) and solidifies at 42oC (gel), doesn’t contribute any nutritive property, it is not hydrolysed by most bacteria and is usually free from growth promoting or growth retarding substances. However, it may be a source of calcium & organic ions. Most commonly, it is used at concentration of 1-3% to make a solid agar medium. New Zealand agar has more gelling capacity than the Japanese agar. Agar is available as fibres (shreds) or as powders.

For preparing agar in Petri plates, 3% agar (by weight) is added to the broth and autoclaved, when the medium is at