Sputum Collection

Introduction on sputum:

Trachaebronchial secretions are often collectively referred to as sputum. Sputum is constituted by plasma, water, electrolytes and mucin. Sputum is viscoelastic, i.e., some of the properties of liquid. Chemical composition revels that sputum is 90% water and only 5% solid. The solid content increases with inflammation. It also slows exfoliation of living cells.

Sputum Collection:

· Before collecting or expectorating sputum the mouth should be prerinsed and this removes contaminants from oral cavity especially.
· For most examinations, a first morning specimen is preferred as it represents the pulmonary secretions accumulated overnight.
· The specimen should be collected in a sterile disposable, impermeable container with screw cap.
· In patients who are uncooperative or cannot produce adequate sputum, induction should be tried.
· Commonly inductants are 10% sodium chloride, acetylcyteine and sterile or distilled water aerosols.

Sputum Culture:

For routine cultures, a loopful of the sputum is inoculated onto one or two blood agar, chocolate agar, MacConkey's agar and thioglycolate broth.

Specimen Collection

A disease can be diagnosed partly by their signs and symptoms. But this does not reveal the full information about the causative organisms and the disease severity. So, to identify the disease causing organism, specimens are collected accordingly. This includes many rules and measures before collection. A specimen must be always collected under aseptic conditions. The different types of specimens and their respective containers are discussed below;

A Quality specimen/sample:

A sample is considered as a good quality sample, when it contains only the infective organisms of the infection and not the environmental contaminations.
A sample collected at a right time of diagnosis of the disease.
A specifically collected sample.
A sample collected before any antibiotic treatment.
A sample collected in a sterile container under aseptic conditions.

Collection of Blood:

The blood consists of a fluid of complicated and variable composition, the plasma, in which suspended erythrocytes, leukocytes and platelets. By using an anticoagulant the formed elements can be separated from plasma. When blood coagulates, the fluid that remains after separation of the clot is serum.

Ways of obtaining blood:

(a) Capillary or peripheral blood:

1. blood can be taken by pricking;
è the lobe of the ear
è the palmer surfaces of the tip of the finger
è in infants, from the plantar surfaces of the heel or the great toe
2. Puncture should be about 3mm deep.
3. An edematous or a congested part should not be used.
4. If the area to be punctured is cold or cyanotic, warm it by massaging or else erroneous may obtain.
5. Clean the site with spirit or alcohols, let dry and puncture.
6. Wipe off the first drop of blood, never press out blood.
7. Having obtained the requisite amount of blood let the patient apply slight pressure over the area with sterile swab.

(b) Venous Blood:

1. Reassure the patient about what is to be done.
2. Inspect the vein, using a tourniquet.
3. Use a syringe of a size according to the amount of blood.
4. Needles of gauze 22 should be used and be 1-1½ inches long.
5. Ask the patient to open and close the first several times.
6. Under aseptic precautions puncture the vein.
7. If it is difficult, first the skin around the vein is punctured and then the vein.
8. Make sure that the bleeding has stopped before the patient leaves.
9. Transfer blood from syringe into the container gently (not through the needle).

Determination of Bacteriocin Activity

To determine the bacteriocin antagonistic activity two methods has been performed. The first method was the plate activity assay and the second was well diffusion method.

(i) Plate Activity Assay:

MRS medium was prepared and plated under aseptic condition. The test organism (Salmonella typhi, E.coli, S.flexinariae) was swabbed over the respective MRS plate eventually. The swabbed plates were kept for 10mins to set. Using sterile inoculation loop, a loop full of direct Leuconostoc mesenteroides culture was touched over the swabbed plate and a small smear was made. The plates were incubated at 37oC for 24hrs.

(ii) Well Diffusion Method:

Mueller Hinton agar medium with 1.5% agar was prepared and plated under aseptic conditions. Using 6mm diameter well cutter, wells were made with equal distance, after the medium was set. A drop of the soft agar was dropped into the well to seal the bottom. The test organism Salmonella typhi, Escherichia coli, and Shigella flexinerrae were swabbed on the respective plates. After allowing for 10mins setting 100μl of the extracted bacteriocin was added into the well. The plates were incubated without inverting, at 37oC for 24hrs. Bacteriocin extracted by both the methods were loaded into their respective wells and checked for its antagonistic activity.

Result

(a) Well diffusion method – zone formed by bacteriocin against Salmonella typhi



(b) Well diffusion method – zone formed by bacteriocin against E.coli



(c) Plate Activity Assay






The bacteriocin was extracted and its antagonistic activity was studied against the indicator organisms by well diffusion method. The zone formed was measured and tabulated:

Salmonella typhi, zone formed – 11mm
Escherichia coli, zone formed – 6mm
Shigella flexinerrae – none

Discussion

Bacteriocin was extracted by cell free supernatant method and the crude supernatant was determined for its antagonistic activity. A well diffusion method was performed. In the activity it was observed that the bacteriocin produced by L.mesenteroides was effective against Salmonella typhi and Escherichia coli. But there was no effect against Shigella flexinerrae. The reviews say that the bacteriocins produced by L.mesenteroides are active against Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Bacillus cereus, L.monocytogenes has been considered as the major food borne pathogen and most activities were against them in food industries. Now these studies reveal the scope for bacteriocins, not only as preservatives but also as an antibiotic for many diseases and infections.

Extraction of Bacteriocin

Bacteriocin extraction from Leuconostoc was done by two methods.

- Bacterial cell lysis method
- Cell free extraction method.

The activity of the supernatant recovered from bacterial cell lysis method was not detectable. It is possible that much greater force is required to disrupt L. mesenteroides, although lactobacillus casei can be disrupted by this method, as shown by Arora et al, Whereas, cell free extraction method showed a great bacteriocin activity against salmonella typhi, and Escherichia coli.

The antibacterial activity appeared to be pronounced between early logarithmic and early stationary phase. Supplementation and/or replacement of nutrients demonstrated that larger quantities of bacteriocin could be produced by addition of yeast extracts (3.0%), NaCl (1.0-2.0%), glucose (1.0%) and tween 80 (0.5%). Maximal activity in composed medium was achieved at initial pH of 5.5 and incubation period of 48hrs at 30 – 37oC.

Procedure:

(i) Bacterial cell lysis method

An overnight culture of Leuconostoc mesenteroides was centrifuged at 10,000 rpm for 20mins at 4oC. The cell was suspended in 100ml of 0.1M phosphate buffer saline (pH 7). The centrifuge and washing was repeated and the cells were pelleted once more. Then, 10ml of PBS and 5g of glass beads were added to the cell pellet and centrifuged at 200rpm and 4oC for 1hr. the suspension was centrifuged and the supernatant was recovered for the determination of antagonistic activity.

(ii) Cell Free Extraction Method

The overnight culture of Leuconostoc mesenteroides was centrifuged at 10,000rpm for 20mins, at 4oC, to obtain a cell-free solution. The supernatant solution pH was adjusted to 7.0 to exclude antimicrobial effect of organic acids. The cell free solution obtained was stirred for 2hrs at 4oC and later centrifuged at 10,000rpm for 1hr at 4oC. The precipitate was resuspended in 25ml of 0.05M potassium phosphate buffer (pH 7.0). The new precipitate was collected and used in the bacteriocin activity assay.

Antimicrobial Protein – Bacteriocin

Bacteriocin is ribosomally synthesized antimicrobial peptides produced by most species of lactic acid bacteria. Most of them are small cationic membrane-active compounds that form pores in the target cells, disrupting membrane potentials and causing death. The production of small cationic peptides with antimicrobial activity is a defense strategy found not only in bacteria, but also in plants and animals. The antagonistic interaction between competing bacteria was described early in 1877, when Pasteur and Joubert noticed that some Escherichia coli strains interfered with the growth of Bacillus anthracis present in infected animals.

Bacteriocins was first detected in 1925 by Andre Gratia, who observed that the growth of some E.coli strains was inhibited by the presence of an antibacterial compound that he called colicin v, released into the medium by E.coli V (virulent strain). Colicin v was later characterized as a heat-stable and dialyzable peptidic compound and in 1954; Pierre Frederic found its genetic determinates in a conjugation – transmissible element similar to the F factor. The antimicrobial peptides produced by bacteria have been grouped into different classes on the basis of the producer organism, molecular size, chemical structure and mode of action.

Bacteriocin Classification:

Jack et. al considered the presence of disulphide and monosulfide (lanthionine) bonds as the basis for their classification as a landmark for their activity spectrum. Accordingly Bacteriocins were classified into four groups:

Antibiotics containing unusual post translationally modified amino acids such as dehydroalanine, dehydrobutirine, lanthionine or B-methyl lanthionine (lantibiotics);
Antibiotics containing at least one disulphide bridge essential for their activity;
Compounds with a single-SH residue that should be in a reduced form for the antibiotic to the active (thiolbiotics); and
Antibiotics without cycteine residues.

According to Klaenhammer, bacteriocins can be classified into four groups on the basis of their molecular mass, thermo stability, enzymatic sensitivity, presence of post translationally modified amino acids and mode of action.

Physical and Chemical characteristics of Bacteriocin:

To perform the bacteriocins lethal activity, it must fulfill two principal requirements:

- to be cationic
- highly hydrophobic

Most bacteriocins are active over a wide pH range (3.0 – 9.0), and while resistance to extreme pH values of 1.0 (acidocin B) and 11.0 (bavaricin A) has been observed. Most of these bacteriocins are cationic at pH 7.0. The high isoelectric point allows them to interact at physiological pH values with the anionic surfaces of bacterial membranes. Heat stability is another major feature of the bacteriocins (stable at 60oc for 120mins at pH 4.5).

They are partially inactivated by autoclaving or heating at 100oC for 120mins. Heat stability was decreased at pH 6.8. Totally inactivated by proteolytic enzymes, pronase, proteinase K, trypsin, and chemotrypsin within 25mins incubation period, whereas treatment with lipase, catalase, and α-amylase did not affect the activity of bacteriocin. The molecular mass was estimated to be 2.5 to 3.0kda based on SDS-PAGE analysis.

Endotoxins

Endotoxin is the lipopolysaccharide (LPS) of the outer membrane of gram negative bacteria. They are bound to bacterium and are released only when the organism lyses since it is a part of cell wall. In contrast to Exotoxin, they are
- heat stable
- toxin only at high dose
- weakly immunogenic
- generally similar, regardless of source
- cannot be toxoided
- Induces many and different pharmacological and immunological changes at low and high concentration.
- At low amounts, elicits a series of reaction : fever, activation of complement by alternative pathway, activation of macrophages and stimulation of B-cells
- In large amounts, it produces shock and hypotension and even death.

Chemistry of Endotoxin:

Bacterial LPS is composed of three parts:

è A glycophospholipid called lipid A
- is a complex array of lipid sources
- Water insoluble since it is hydrophobic
- Responsible for toxicity
- Even when paired with artificial carriers, its activity is restored.

è A Core Polysaccharide with ethanolamine and phosphate
- Common to all gram negative
- serve as carrier

è ‘O’ Antigen
- A long side chain of species specific
- Unusual polysaccharide
- serve as carriers for lipid A

Major effects of Endotoxin:

- At low concentration it sets of series of alarm reactions.
- At high range, it induces shock.
- Overlapping of these complex events depends on the amount of Endotoxin, route of infection and previous exposure of host to those substances.
- The primary target cells of Endotoxin are mono-nuclear phagocytes (monocytes, macrophages of spleen, bone marrow, lung alveoli, peritoneal cavity and kuffer cells), neutrophils, platelets and B- lymphocytes since they have specific Endotoxin receptor.

Pharmacological toxins

Pharmacological toxins – Function by elevating or depressing normal cell functions but which do not result in death of their target cell.

Toxins that elevate cyclic AMP – Cholera toxin

These toxins raise the concentration of cyclic AMP (cAMP) without damaging the cell. The excess cAMP inhibits chemotaxis and phagocytosis, thus reducing their power to kill microorganisms. cAMP can be increased in several ways,
- some pathogens pour out cAMP themselves
- some secrete adenyl cyclase to make more cAMP from ATP
- secrete toxin that alters the activity of adenyl cyclase of host cells.

Cholera toxins (Best example)

It is an enterotoxin, a protein of mol.wt 90,000. Target tissue is the epithelium of the small intestine. It has separate A and B subunits
B component has specific affinity for the intestinal epithelial mucosa via gangliosiodic receptor.
A subunit has affinity to ADP-ribosylate of the target protein.
The target protein is part of a complex that makes cAMP. Synthesis of cAMP becomes unregulated and is made in large amount. This provokes loss of fluid and copious diarrhea which is the characteristic of Cholera.

Synthesis of cAMP

- The cyclic AMP is synthesized by the enzyme adenylate cyclase.
- It is composed of 3 proteins - Gs, R and cyclic itself.
- Gs protein is a GTP binding protein that has 2 conformational states.
- When it binds GTP, it stimulates adenylate cyclase to make cAMP.
- This effect is normally of short duration because Gs protein is also a GTPase that cleaves GTP to GDP.
- The activity of adenyl cyclase is thus determined by the balance of binding and hydrolysis of GTP by Gs protein.
- The balance of binding of GTP by Gs proteins is determined by R-protein which stimulates Gs.
- R-protein is a receptor for one of several hormones (adrenergic).
- Hence when R-protein binds one of these hormones, it interacts with Gs protein to increase its binding of GDP.
- Gs protein becomes active to stimulate adenyl cyclase.

Toxin - Mode of action

The A domain remains in the latent form even after uptake, gets activated only by proteolytic cleavage and reduction of disulfide bond. All form of these toxins have common mode of action - Catalyze transfer of the ADP group from the coenzyme.

Diphtheria toxin

- Its a protein of about 62,000 mol.wt
- The A and B domains are synthesized as single polypeptide chain
- The AB dimeric domain undergoes endocytosis and membrane translocation (out of vesicles with cytoplasm) to release domain A to reach cytoplasm and begin its toxic action.
- Domain A is resistant to denaturizing and is long-lived inside the cell.
- A single molecule can kill a cell
- Killing takes place by catalyzing ADP-ribosylation (addition of ADP-ribose group) to the eukaryotic elongation factor 2(EF2).
- EF2 is a protein that catalyzes the hydrolysis of GTP, which drives the movement of ribosome on eukaryotic mRNA.
- The substance for this reaction is the coenzyme NAD.

NAD+ + EF-2 =======>> ADP-ribosyl-EF2 + Nicotinamide

- The modified EF2 protein cannot participate in the elongation chain of protein synthesis and the cell dies because it can no longer synthesis protein.
- EF2 is the only known substrate for diphtheria toxin, and the specificity is that EF2 contains a rare modification in one of its histidine residues and this site is recognized by diphtheria toxin for ADP-ribosylation.
- Mutant cells cannot modify histidine and become resistant to the toxin.
- This leads to myocarditis, neuritis, necrosis of mucous membrane.

Mode of entry into the cell - Receptor mediated endocytosis

Dimeric exotoxin binds to a receptor-ligand complex that is internalized in a clathrin-coated pit that pinches off to become a coated vesicle. By the time, the molecule A and B are cleaved at protease sensitive site, but remains covalantly associated. Clathrin coat depolymerizes resulting in an uncoated vesicle (endosome). pH in the endosome decreases owing to the AT activity by reduction of disulphide bond. Low pH causes A and B component to separate, which is called as CURL ( Compartment of Uncoupling of Receptor and Ligand ). B domain is then recycled to cell surface. A domain moves through the cytosol, and inhibits protein synthesis.

Toxins - A Brief Introduction

Toxins are biological weapons and a specific soluble metabolic product of microorganisms directed at us, which causes deleterious effect on host. It is produced by Bacteria, Fungi, Protozoa and Worms. Their mode of action paves way to understand the pathophysiology of infection. Toxemia refers to the condition caused by toxins that have entered the blood of host.

Types of toxins – Exotoxin, Endotoxin.

Exotoxins

- Synthesized by specific pathogens that often has plasmids or prophages bearing the Exotoxin gene (mainly Gram +, sometimes Gram -). Ex: E.Coli, Vibrio, Shigella.
- Soluble protein, usually released in the surrounding as the pathogens grow.
- In many instances, they travel far from the site of infection to other target cells.
- Sometimes bound to Bacterial surfaces, released on cell lysis.
- Heat-labile proteins inactivated at 60-80c
- Mostly lethal – 1gm of tetanus, botulium or Shigella toxin is enough to kill 10 million people.
- Highly immunogenic and stimulate the production of neutralizing antibodies (antitoxins).
- Easily inactivated by formaldehyde, iodine to form immunogenic toxoids.
- Usually unable to produce fever.
- Categorized as neurotoxins, cytotoxins or enterotoxins based on their mechanism of action.

Structural Model

It occurs in many forms, the general structural model to which they frequently conform is AB model.
‘A’ Subunit – an enzymatic subunit, responsible for toxic effect in host cell
‘B’ Subunit – binding subunit, binds to target cells but non toxic and biologically inactive. Binds with specific receptors on target cells (or) tissue such as sialogangliosides.
Ex: Gm1 for Cholera toxin, GT1/GD1 for Tetanus toxin, GD1 for Botulinum toxin.

Entry of Subunits

Several mechanisms are proposed by the toxins. In one mechanism B subunit inserts into the membrane and creates a pore for the A subunit to enter. In another, entry is by receptor mediated endocytosis. The mode of entry is explained in the diagram given above.

Isolation & Characterization Of Leuconostoc Mesenteroides From Cheese – Part 3

RESULTS AND DISCUSSION:
Isolation of Leuconostoc:

From the cheddar cheese, two types of colonies were isolated. One colony was yellow colored shiny, smooth and entire marginated. And the other was pale white, slimy, smooth colonies. Gram staining results showed: 1) gram positive bacilli, 2) gram positive cocci, and 3) gram positive coccobacilli. So to isolate Leuconostoc from other LAB organisms, vancomycin was added to the MRS medium.

Characterization of Leuconostoc:
Cultural & Biochemical characteristics - Results
Gram’s staining Gram positive, coccobacilli
Motility Motile
Capsule staining Non-capsulated
Spore staining Non-sporing
Growth at 37oC +
Growth at 30oC +
Growth at 4oC +

On MRS medium:
At 37oC Pale yellow colored, smooth, slimy,
entire marginated, convex colonies.

At 4oC In refrigeration, colonies turned white,
and slow growth was observed.

On MacConkey agar medium Lactose fermenting, pink colored
colonies.

On blood agar medium Non-hemolytic, colorless colonies.

Biochemical tests:
Indole -
Methyl red -
Voges Proskauer +
Citrate -
Urease -
Arginine -

Carbohydrate fermentation tests:
Glucose + (acid and gas)
Arabinose +
Lactose +
Mannitol -
Sucrose +
Dextrose +
Maltose +

Discussion:

From the three different colonies isolated from cheese (fig-1), Leuconostoc was isolated separately by its specific vancomycin resistant character (fig-2). Species was identified by its biochemical and carbohydrate tests. First, Leuconostoc mesenteroides was differentiated on the basis of Arginine hydrolysis. Only L.cremoris and L.mesenteroides is Arginine hydrolyser and both are possible in cheese. Secondly, Arabinose fermenters. Between both the species, only L.mesenteroides is Arabinose fermenters (Bettache Guessas and Mebrouk Kihal, 2004). Thus the species was confirmed as Leuconostoc mesenteroides.

Isolation and Characterization of Leuconostoc Mesenteroides from Cheese – Part 2

Materials & Methods

1. Sample collection - Cheddar cheese & Mozzarella cheese
2. Serial dilution and spread plate method
3. Isolation of Lactic acid bacteria in MRS medium
4. Selective Isolation of Leuconostoc from other LAB
5. Addition of vancomycin to MRS medium
6. Leuconostoc was isolated and maintained at 4oC
7. Species identification

Biochemical characterization

Sample collection: Cheddar cheese and Mozzarella cheese used in their study were obtained from retail super market (Nilgiris), Chennai. A well vacuum packed cheese was obtained.

Bacterial strains and culture:

Isolation of Leuconostoc:

Culture Media: Leuconostoc are fastidious chemo-organotrophic bacteria. Therefore, culture media must be rich in nutrients such as carbohydrates as energy providers, amino acids as nitrogen compounds, salts and vitamins. The most widely used medium was MRS medium (Man Rogosa Sharpe). The supplements studied in MRS medium are glucose, peptone, yeast extract, beef extract, dipottasium hydrogen phosphate, ammonium citrate, magnesium sulphate, manganese sulphate sodium acetate, and tween 80. Tween 80 usually increases the growth of Leuconostoc by providing oleic acid incorporated into the cell membrane.

Composition of MRS medium

Media gm/lit

Glucose 1.0gm
Peptone 1.0gm
Beef extract 0.8gm
Yeast extracts 0.5gm
Di-potassium hydrogen phosphate
(K2HPo4) 0.2gm
Magnesium sulphate (MgSo4) 0.2gm
Manganese sulphate (MnSo4) 0.005gm
Ammonium citrate 0.2gm
Sodium acetate 0.5gm
Tween 80 0.1gm
Agar 2.0gm
pH 6.8gm

The medium was sterilized at 121oC for 15mins. Leuconostoc was enumerated by spread plate method. Serial dilutions were made from the master dilutions containing 1gm of cheddar cheese in 99ml of distilled water which gives a dilution of 10-1 (serial dilutions were made upto 10-1). 0.1ml of each dilution was spreaded on MRS medium, and incubated at 37oC for 24hrs.

From food sample, all LAB are counted since the complex nutrient requirements and optimal conditions for the growth are roughly comparable for lactobacillus, Pediococcus and Leuconostoc. So, for selective isolation of Leuconostoc from other lactic acid bacteria (vancomycin sensitive), 30mg/ml of vancomycin solution was added to the MRS medium. The Leuconostoc strain isolated was maintained by sub-culturing in MRS medium.

Indicator bacterial strains:

Salmonella typhi, ATCC-21563
Escherichia coli, ATCC-1023
Shigella flexinerrae, ATCC-11461

were received from NCIM and maintained by sub-culturing in nutrient agar slants for further biochemical tests. The incubation period for the test organisms was 37oC for 24hrs. All the cultures were stored at 4oC.

Media used for cultural characterization of Leuconostoc:

MacConkey agar medium
Blood agar medium

Biochemical characterization of Leuconostoc for species identification:

Gram’s staining
Motility test – hanging drop method
Capsule staining – negative staining
Spore staining – Scaffer-fulton method

Biochemical tests:

Indole
Methyl red
Voges Proskauer
Citrate
Nitrate
Urease
Arginine hydrolysis

Carbohydrate fermentation test:

Glucose
Arabinose
Fructose
Sucrose
Maltose
Galactose
Lactose
Mannital

All the above biochemical tests were performed and the results were noted to differentiate Leuconostoc species. The differentiating test among Leuconostoc species are catalase test, arginine test and carbohydrate fermentation tests.

Separate biochemical test were performed for the indicator organisms to confirm the strains and results were noted.

Isolation and Characterization of Leuconostoc Meseteroides from Cheese – Part 1

Leuconostoc:

This genus was called as Betacoccus by Oria – Jensen. It contains the heterofermentative lactic bacteria, which ferments sugars to produce 50% lactic acid, plus, 25% acetic acid and ethyl alcohol and 25% carbon dioxide. These other compounds are important as they impart particular tastes and aromas to the final product. The major subsps of Leuconostoc are,

Leuconostoc mesenteroides
Leuconostoc dextranicum
Leuconostoc cremoris
Leuconostoc lactis

Sources Of Leuconostic:

Leuconostoc are used as mesophilic starters in combination with other starters for making cottage and cream cheese, cultured milk, buttermilk. It produces acetic acid from citrated and may be added to cottage cheese to prevent spoilage by slime producing Pseudomonades. Leuconostoc are also found in milk and milk products, vegetables, plant surfaces. Dextrans produced by Leuconostoc mesenteroides are used as stabilizers in ice cream mixes.

Special Focus On Leuconostoc MesenteroidesS:

Leuconostoc mesenteroides differs from other lactic acid species as it can tolerate fairly high concentration of salt and sugars (unto 50% sugar). Leuconostoc mesenteroides initiate growth in vegetables more rapidly over a range of temperatures and salt concentration than any other lactic acid bacteria. It produces carbon dioxide and acids which rapidly lower the pH and inhibit the development of undesirable microorganisms. The carbon dioxide produced replaces the oxygen, making the environment anaerobic and suitable for the growth of subsequent species of lactobacillus and inhibits the growth of aerobic micro flora. Removal of oxygen also helps to preserve the color of vegetables and stabilizes any ascorbic acid that is present.

These characters of Leuconostoc mesenteroides made me to focus on its isolation and characterization, further more antimicrobial activity, bacteriocin.

Growth Requirements

Leuconostoc have a complex growth requirement with an optimum temperature of 20-30°C. They are fastidious chemo organotrophic bacteria requiring rich nutrient sources such as carbohydrates, nitrogen sources, salts, vitamins and sugars. The medium used for the isolation and enumeration of Leuconostoc was MRS medium (Man Rogosa Sharpe Medium). MRS medium contains all the essential substrates for the growth of Leuconostoc.

In isolating Leuconostoc from nature or food samples, the combination of the presence of agents inhibitory to most lactic acid bacteria other than Leuconostoc spp and the agents stimulatory for Leuconostoc spp in the medium selects for Leuconostoc spp.

The vancomycin resistant character of Leuconostoc spp. favored for the isolation from cheese sample. Other lactic acid bacteria are vancomycin sensitive. MRS medium containing 30mg/ml concentration of vancomycin was used as the special medium for Leuconostoc isolation. Comprehensive reviews of Leuconostoc differential and selective medium have been published by Garvie, Tenber & Gets and Cogan.

My first blog in microbiology

I’ve completed M.Sc., in Microbiology and I would like to share all my knowledge and experiences in my field of biology. Hope this blog will help the upcoming students and teachers too…