Microgen Culture Media

DEHYDRATED CULTURE MEDIA

culture media products are made of high quality ingredients so as to produce maximum good results as expected by our customers. It is of great significance to know the functions and reactions of a particular ingredient and its interaction with other ingredients in a media. Here arises the necessity of strict evaluation of the ingredients.

In each and every ingredient undergoes thorough testing prior to manufacturing by our well-equiped and highly efficient quality control department. Maximum quality control measures are taken throughout the manufacturing process to give the desired results. It will be our greatest effort to provide products of maximum attainable quality to our customers.

We serve a wide range of culture media products to meet the var ious requirements of Dairy , Pharma ceuticals , Veterinary, Cosmetics, Water Treatment, Public Health and Medical Microbiology departments.

Microorganism growth on culture media depends on a number of important factors :

• Proper nutrients must be available.
• Oxygen or other gases must be available as required.
• Moisture is necessary.
• The medium must have an appropriate pH.
• Proper temperature relations must prevail.
• The medium must be free of interfering bioburden.
• Contamination must be prevented.

A satisfactory microbiological culture medium must contain available sources of

• Carbon,
• Nitrogen,
• Inorganic phosphate and sulfur,
• Trace metals,
• Water,
• Vitamins

These were originally supplied in the form of meat infusion, Beef or yeast extracts frequently replace meat infusion in culture media. The addition of peptones, which are digests of proteins, provides readily available sources of nitrogen and carbon.

The pH of the culture medium is important for microorganism growth. Temperature is another important parameter mesophilic bacteria and fungi have optimal growth at temperatures of 25-40 C; thermophilic bacteria ("heat loving") grow only at temperature greater than 45 C psychrophilic ("cold loving") organisms require temperatures below 20 C. Human pathogenic organisms are generally mesophiles.

COMMON MEDIA CONSTITUENTS
Media formulations are developed on the ability of bacteria to use media components.

CONSTITUENTS	SOURCE
Amino-Nitrogen	Peptone, protein hydrolysate, infusions and extracts
Growth Factors	Blood, serum, yeast extract or vitamins, NAD
Energy Sources	Sugar, alcohols and carbohydrates
Buffer Salts	Phosphates, acetates and citrates
Mineral Salts and Metals	Phosphate, sulfate, magnesium, calcium, iron
Selective Agents	Chemicals, antimicrobials and dyes
Indicator Dyes	Phenol red, neutral red
Gelling agents	Agar, gelatin, alginate, silica gel

Media Ingredients
Peptone, protein hydrolysates, Infusions and extracts are the major sources of nitrogen and vitamins in culture media. Peptones are water-soluble ingredients derived from proteins by hydrolysis or digestion of the source material, e.g. meat, milk.

Carbohydrates are employed in culture media as energy sources and may be used for differentiating genera and idenifying species.

Buffers maintain the pH of culture media.

Selective Agents include Bile Salts, dyes and antimicrobial agents.

Bile Salts and desoxycholate are selective for the isolation of gram-negative microorganisms, inhibiting gram-positive cocci.

Dyes and indicators are essential in the preparation of differential and selective culture media. In these formulations, dyes act as bacteriostatic agents, inhibitors of growth or indicators of changes in acidity or alkalinity of the substrate.

Antimicrobial agents are used in media to inhibit the growth of bacteria, yeasts and fungi.

Solidifying agents, including agar, gelatin and albumin, can be added to a liquid medium in order to change the consistency to a solid or semisolid state.

ENVIRONMENTAL FACTORS IN CULTURE MEDIA
Atmosphere
Most becteria are capable of growth under ordinary conditions of oxygen tension. Obligate aerobes require the free admission of oxygen, while anaerobes grow only in the absence of atmospheric oxygen. Between these two groups are the microaerophiles, which develop best under partial anacrobic conditions, and the facultative anaerobes, which are capable of growing in the presence or absence of oxygen. Anaerobic conditions for growth of microorganisms are obtained in a number of ways :

• Addition of small amounts of agar to liquid media;
• Addition of fresh tissue to the medium;
• Addition of a reducing substance to the medium, e.g., sodium thioglycollate, thioglycollic acid and L-cystine;
• Displacement of the air by carbon dioxide;
• Absorption of the oxygen by chemicals;
• Inoculation into the deep layers of solid media or under a layer of oil in liquid media.

Many microorganisms require an environment of 5-10% CO₂. Levels greater than 10% are often inhibitory due to a decrease in pH as carbonic acid forms. Culture media vary in their susceptibility to form toxic oxidation products if exposed to light and air.

Water Activity
Proper moisture conditions are necessary for continued luxuriant growth of microorganisms. Organisms require an aqueous environment and must have "free" water. "Free" water is not bound in complex structure and is necessary for transfer of nutrients and toxic waste products. Evaporation during incubation or storage results in loss of "free" water and reduction of colony size or total inhibition of organism growth.

Protective Agents and Growth Factors
Calcium carbonate, soluble starch and charcoal are examples of protective agents used in culture media to neutralize and absorb toxic metabolites produced by bacterial growth.

NAD (V factor) and hemin (X factor) are growth factors required by certain bacteria; e.g., Haemophilus species and for enhanced growth of Neisseria species.

Surfactants, including Polysorbate 80, lower the interfacial tension around bacteria suspended in the medium. This activity permits more rapid entry of desired compounds into the bacterial cell and can increase bacterial growth.

The preparation of culture media from dehydrated media requires accuracy and attention to preparation. The following points are included to aid the user in successful and reproducible preparation of culture media.

DEHYDRATED MEDIA AND INGREDIENTS

• Store in a cool (15-300C), dark and dry area unless otherwise specified.
• Note date opened.
• Check expiry (applied to intact container).
• Verify that the physical characteristics of the powder are typical.

GLASSWARE / PLASTICWARE

• Use high quality, low alkali borosilicate glass.
• Avoid detergent residue.
• Check for alkali or acid residue with a few drops of bromo thymol blue pH indicator (yellow is acidic; blue is alkaline).
• Use vessels at least 2-3 times the volume of medium.
• Do not used etched glassware.

EQUIPMENT

• Use measuring devices, scales, pH meters, autoclaves and other equipment that are frequently and accurately calibrated.

WATER

• Use distilled or deionized water.
• pH 5.5-7.5.

DISSOLVING THE MEDIUM

• Accurately weigh the appropriate amount of dehydrated medium.
• Dissolve the medium completely.
• Agitate the medium while dissolving.
• Take care to not overheat. Note media that are very sensitive to overheating. Overheated media will frequently appear darker. Do not heat in a microwave.

STERILIZATION

• The autoclave set-temperature should be 1210C.
• Routine autoclave maintenance is important. Ask manufacturer to check for "hot" and "cold" spots.
• The recommended 15 minute sterilization assumes a volume of 1 liter or less. Larger volumes may require longer cycles. Check with your autoclave manufacturer for recommended load configurations.
• Quantities of media in excess of two liters may require an extended autoclave time to achieve sterilization. Longer sterilization cycles can cause nutrient concentration changes and generation of inhibitory substances.

ADDING ENRICHMENTS AND SUPPLEMENTS

• Enrichments and supplements tend to be heat sensitive.
• Cool medium to 45-550C in a waterbath prior to adding enrichments or supplements.

pH

• Commercial dehydrated media are designed to fall within the specified pH range after steam sterilization. The pH tends to fall approximately 0.2 units during steam sterilization.
• For filter sterilization, adjust the pH, if necessary, prior to filtering.
• Avoid excessive pH adjustment.

DISPENSING MEDIA

• Ensure gentle mixing during despensing.
• Cool the medium to 50-550C prior to dispensing to reduce water evaporation.
• Dispense quickly.
• If using an automatic plate dispenser, dispense general purpose media beforedispensing selective media.
• Immediately recover or recap tubes to reduce the chance of contamination. Leave Petri dish covers slightly open for 1-2 hours to obtain a dry surface.

STORAGE AND EXPIRY

• In general, store steam-sterilized plated media inverted in a plastic bag or other container in a dark refrigerator for up to 1-2 weeks.

QUALITY CONTROL

• For media prepared in-house, each lot of every medium must be tested.
• Maintain Quality Control Organisms appropriately.
• Maintain appropriate records.
• Report deficiencies to the manufacturer.

REFERENCES

1. Block, S. 1992. Sterilization, p. 87-103. Encyclopedia of microbiology, vol. 4. Academic Press, Inc., San Diego, CA.
2. Cote, R. J., and R. L. Gherna, 1994. Nutrition and media,p. 155-178. In P. Gerhardt, R.G. E. Murray, W.A. Wood, and N.R. Krieg (ed.), Methods for general and molecular bacteriology. American Society for Microbiology, Washington, D.C.
3. The United States Pharmacopels (USP XXIII) and The National Formulary (NF 18). 1995. Sterilization and sterility assurance of compendial articles, p. 1976-1980. United States Pharmacopeial Convention Inc., Rockville, MD.
4. Perkins, J.J. 1969. Principles and methods of sterilization in health sciences, 2nd ed. Charles C. Thomas, Springfield, IL.
5. Leahy, T.J. 1986. Microbiology of sterilization processes. In F.J. Carleton and J.P. Agalloco (ed.), Validation of aseptic pharmaceutical processes. Marcel Dekker, Inc. New Yark, N.Y.
6. Simko, R.J. 1986, Organizing for validation. In F.J. Carleton and J.P. Agalloco (ed.), Validation of aseptic pharmaceutical processes. Marcel Dekker, Inc. New York, N.Y.

BACTERIA CONTROL STRAIN SOURCE
An integral part of quality control testing include quality control organisms. Microorganisms should be obtained from reputable sources, for example, the American Type Culture Collection (ATCC ) or other commercial sources.

MAINTENANCE / FROZEN STOCK CULTURES
If using commercial stock cultures, follow the manufacturer's recommendations for growth and maintenance.

To prepare frozen stock cultures of Staphylococcus species, Streptococcus species, Enterobacteriaceae and Pseudomonas aeruginosa :

1. Reconstitute the stock culture, if necessary.
2. Inoculate multiple plates of a general purpose medium (e.g., TSA or blood agar).
3. Incubate plates for 18-24 hours in an appropriate atmosphere and at the recommended temperature.
4. Check for purity and correct colony morphology.
5. If necessary, verify biochemical tests.
6. Remove sufficient growth from a confluent area to prepare a 0.5 McFarland standard (1-2 x 10 CFU/ml).
   For fastidious organisms, adjust to a 1.0 McFarland.
7. Suspend the growth in 50-100 ml of cryoprotective medium, e.g., Tryptic Soy Broth with 10-15% Glycerol, Skim Milk or sterile defibrinated sheep blood.
8. Dispense 0.5-1.0 ml into sterile glass plastic freezing vials, Prepare enough vials for one year of storage.
   Assume only one freeze/thaw cycle per vial. Assume at least one fresh culture every four weeks.
9. Store vials at or below -500C (freezer) for one year.
   Organisms will keep longer (indefinitely) if stored in ultra low temperature freezer or in a liquid nitrogen tank.

TO USE A FROZEN CULUTRE :

1. Thaw the vial quickly.
2. Use the culture directly subcutlure.
3. Discard any unused cell suspension.

WORKING CULTURES
Prepare no more than three serial subcultures from a frozen stock culture.

1. Inoculate an agar slant or plate with the frozen stock culture and incubate overnight.
2. Store the working culture at 2-8⁰C or at room temperature for up to four weeks.
3. Check for purity and appropriate colony morphology.

or

1. Use the frozen stock culture directly as a working culture.

Maintain anaerobic cultures in Cooked Meat Medium or another suitable anaerobic medium. Alternatively, use frozen anaerobic cultures.

TEST PROCEDURE

1. Inoculate an agar plate from the "working culture".
2. Incubate overnight.
3. Suspend 3-5 isolated colonies with typical appearance in a small volume (0.5-1.0 ml) of TSB. Incubate 4-5 hours in an appropriate atmosphere and temperature.
4. Adjust the turbidity to 0.5 McFarland and 0.08-0.1 absorbance units at 625 nm.

or

1. Adjust an overnight culture to a 0.5 McFarland.
2. Plate 0.01 ml of the specimen to confirm a colony count of 1-2 x 10 CFU/ml. If using a frozen culture, confirm the appropriate density.

TO TEST CULTURAL RESPONSE
NON-SELECTIVE MEDIA
Dilute the cell suspension 1:100 in normal saline or purified water. Inoculate each plate with 0.01 ml to give 1-2 x 10 CFU/plate. Reduce the inoculum ten fold, if necessary, to obtain isolated colonies.


SELECTIVE MEDIA AND TUBED MEDIA
Dilute the cell suspension 1:10 in normal saline or purified water. Streak each plate with 10.01 ml of the suspension to provide 1-2 x 10 CFU/plate. Reduce the inoculum ten fold, if necessary, to avoid overwhelming some selective media.

RESULTS
For general-purpose media, sufficient, characteristic growth and typical colony morphology should be obtained with all test strains. For selective media, growth of designated organisms is inhibited and adequate growth of desired organisms is obtained. Color and hemolytic reaction criteria must be met.

REFERENCE
National Committee for Clinical Laboratory Standards. 1996. Quality assurance for commercially prepared microbiological culture media, 2nd ed. Approved standard. M22-A-2, vol. 16, no. 16. National Committee for Clinical Laboratory Standards, Wayne, PA.

"Typical" chemical compositions have been determined on media ingredients. The typical analysis is used to select products for research or production needs when specific nutritional characteristics are required. The specifications for the typical analysis include :

• Physical characteristics,
• Nitrogen content,
• Amino acids,
• Inorganics,
• Vitamins, and
• Biological testing.

All values are presented as weight/weight; % = g/100 g.

GLOSSARY

ASH
The higher the ash content, the lower the clarity of the prepared ingredient. The ash content includes sodium chloride, sulfate, phosphates, silicates and metal oxides. Acid-insoluble ash is typically from silicates found in animal fodder.

MOISTURE
Lower moisture levels (<.5%) are preferred. Higher moisture levels in dehydrated ingredients may reduce stability. In the presence of high moisture and high ambient temperatures, chemical interactions will cause darkening of the product and falling pH. These characteristics indicate product deterioration.

NITROGEN
Total Nitrogen : Total nitrogen is usually measured by the kjeldhal digestion or titration method. Not all organic nitrogen is nutritive. Percent (%) nitrogen x 6.25 ~ % proteins, peptides or amino acids present.
Amino Nitrogen : The amino nitrogen value shows the extent of protein hydrolysis by measuring the increase in free amino groups. This is a nutritionally meaningful value.

PH
Changes in pH from specified values, either after storage or processing, indicate deterioration. These changes are uaually accompanied by darkening of the end product. Hydrolysates vary in their pH resistance according to their inherent buffering (phosphate) capacity.

PHOSPHATES
High-phosphate ingredients may be unsuitable for pH indicator media due to the inherent buffering of phosphates.

However, phosphates do aid in gas production, which can be enhanced by deliberate addition of sodium phosphate.

SODIUM CHLORIDE
The NaCl content may reflect significant pH adjustments during processing e.g., acid hydrolysates. (See Ash).

TRACE METALS
Trace metals can directly antagonize antimicrobial activity in vitro or impact toxin production, chelating agents may be added to culture media to sequester trace metals and clarify the media.

The Dehydrated Media are highly hygroscopic and get deteriorated easily unless stored in a cool & dry place away from bright light.

So care must be taken in the storage of dehydrated media.

Following instructions should be followed while using dehydrated culture media.
1. Read carefully the instruction given on the label.
2. Note the batch no. and date before opening the container.
3. Before use confirm that the media is not deteriorated physically.
4. Since the media is hygroscopic in nature, please ensure that the container is tightly closed and stored in cool and dry place after use.

DISSOLVING THE MEDIA : For dissolution use clean undamaged glassware two-three times larger in volume than the final volume of media to be prepared. Water which meets the U.S.P./I.P specifications should only be used.
Place accurately weighed amount of the medium in a clean dry flask. Add part of water and swirl until dissolved then add the remaining water through the sides of the flask to make
up the required volume. For complete dissolution heat the media taking care to avoid overheating or scorching.

ADJUSTMENT OF pH : After preparations the pH of the media should be in concordance with value mentioned on the label at 25°C. If required, the pH should be adjusted to the
specified value by adding 1N or 0.1N HCI or NaOH solution.

STERILIZATION : Sterilization of the media is usually carried out at 121 °C for 15 minutes using an autoclave. Autoclave efficiency should be checked from time to time.

POURING OF STERILIZED MEDIA : After sterilization Agar media should be poured into petri dishes at 45-50°C. The medium should be mixed well avoiding bubble formation.
Agar surface should be dried at 30-40°C in the incubator before inoculation to avoid microbial swarming.

STORAGE OF PREPARED MEDIA : Unless the medium is not used in the same day it is prepared, then it should be kept in moisture proof containers.

Agar containing media should not be stored at higher temperatures. Agar plates should be stored at 2-8°C in sealed moisture proof containers. Stability of the prepared culture
media is limited and varies considerably. Never store media below 0°C as it destroys its gel strength.

DISPOSAL OF MEDIA : All specimens and cultures should be carefully handled and must not disposed without autoclaving. Cultures in vessels should be autoclaved for
approximately 30 minutes at 121 °C before disposal.

STORAGE OF MICROGEN CULTURE MEDIA PRODUCTS : For obtaining desired results style="margin: 0px 6px" src="images/micrigen-logo1.png"> culture media products should be stored in specified storing conditions. It is
recommended to use the products in the order of Batch number/Mfg. date.

Storage temperature and shelf-life of the style="margin: 0px 6px" src="images/micrigen-logo1.png"> culture media products are as follows.

(a) Dehydrated media : Dehydrated media if stored under specified conditions will have shelf-life of 2 to 5 years. Storage temperature for dehydrated media are preferably
between 8 and 25°C.

(b) Selective supplements : Except Horse serum (-20°C) all other supplements are required to store at 2 - 8°C. These products have a shelf-life of 1 - 3 years.

(c) Antimicrobial susceptibility discs : These are to be stored at -20°C but working stock at 28°C Shelf-life is from 1 to 3 years.

certain precautions while using dehydrated media

Faults	Reasons
Drift in pH hydrolysis of	Overheating, incomplete mixing, prolonged sterilization, use of alkaline glass, impure water, ingredients, prolonged storage at high temperature.
Incomplete Solubility	Inadequate heating of agar media incomplete mixing.
Darkening	Overheating of the medium, excess amount of dehydrated powder, improper mixing.
Soft Gel	Agar not in solution, improper reconstitution of dehydrated medium, acid hydrolysis of agar, failure to compensate for dilution of agar by the incoclum.
Loss of Growth differentiating properties	Promoting of repeated remelting, excessive heating, incomplete mixing, failure to compensate for dilution of ingredients, disturbance in the formula by inoculum carriers, etc.
Abnormal Colour of Medium	Deteriorated, dehydrated medium, improperly washed glassware, impure water.
Contamination	Improper/Insufficient sterilization. Poor technique in adding enrichments and pouring plates.