Dry nutrient media are highly hygroscopic and should be stored in a cool, dry place away from bright light. These media are intended for laboratory use only. Read the instructions on the label carefully. Pay special attention to the composition of the medium, instructions for its preparation and use. Also pay attention to the expiration date and batch number. Before use, make sure that the medium has not changed its physical properties and the shelf life has not expired.

Nutrient media tend to form lumps (clumping) under the following conditions:

• With high humidity during storage;
• If the container has been open for a long time;
• If the container is constantly closed loosely;
• If the environment is very old.

To prepare the medium, use clean, intact glassware and distilled or deionized water that meets the requirements of the US Pharmacopoeia and the International Pharmacopoeia for clean water. Place an aliquot of the medium in a clean, dry flask, the volume of which is 2-3 times the final volume of the finished medium. Add part of the prepared amount of water and stir with rotary motions until dissolved.

Then little by little, along the wall of the flask, add the remaining amount of water. Most broths at this stage are completely dissolved and appear clear. For complete dissolution, use an open flame, a hot stove or boiling water, avoiding excessive heating and burning of the medium.

Normally, dry media reconstituted with distilled or deionized water at 25°C have the pH values ​​indicated on the label. However, it is recommended, especially when using media that have been stored for a long time, to check and, if necessary, correct the pH value.

Measurement of pH in liquid media should be carried out at 25°C, and in molten dense media at 40-45°C. The pH value is adjusted to the required value by adding 1 N or 0.1 N solutions of hydrochloric acid or sodium hydroxide to a certain volume of the sample (for example, to 50 or 100 ml of medium). After recalculation, the required amount of acid or alkali is added to the remaining volume of the medium.

Sterilization

The fully prepared medium is sterilized as indicated on the label.

Sterilization is usually carried out in an autoclave for 15 minutes at 121 ° C. The ratio of temperatures and pressures is given in Table 1.

Table 1. Correlation of temperatures and pressures

From time to time it is necessary to check the effectiveness of autoclaving. The temperature conditions in different points of the working chamber of the autoclave are not the same. They can be checked with the help of two vials with a 2% solution of glucose in a 2% solution of sodium hydrogen phosphate, which are placed in different places of the working chamber. Heating gives the solution a brown color. The contents of the vial located near the steam inlet have a more intense brown color than the contents of the other vials.

From time to time it is necessary to check the effectiveness of autoclaving. The temperature conditions in different points of the working chamber of the autoclave are not the same. They can be checked with the help of two vials with a 2% solution of glucose in a 2% solution of sodium hydrogen phosphate, which are placed in different places of the working chamber. Heating gives the solution a brown color. The contents of the vial located near the steam inlet have a more intense brown color than the contents of the other vials.

All autoclaves must be regularly checked for efficiency and effectiveness.

Physical control parameters are temperature and steam pressure. It is also necessary to check the saturation of the steam and the safety of the operation of the valves. In Russia and some other countries, the autoclave control program includes biological tests that demonstrate the effect of sterilization.

Physico-chemical tests show whether the specified temperature was reached during sterilization, and some of them also show the adequacy of exposure at a given temperature.

High temperatures and prolonged heating are a common cause of a shift in pH, darkening of nutrient media, precipitation, poor gelation, and loss of quality of the nutrient medium as a whole. It is assumed that all sterilized media are in the form of a solution, otherwise darkening of the media is possible (Maillard reaction).

1. In order to avoid the appearance of abundant condensate on the lids of the cups, sterile agar media must have a temperature of 45-50°C before pouring. The medium should be thoroughly mixed, preventing the formation of air bubbles, aseptically poured into Petri dishes. Before sowing, the surface of the agar media should be dried in aseptic conditions at 30-40 ° C in a thermostat.
2. Addition of blood: It is better to use defibrinated blood than anticoagulated blood for blood agar preparation. It is better to use as much fresh blood as possible, and if it was stored (at + 2 … 8 ° C, without freezing), then it is preheated to 35-37 ° C in a thermostat, and then added to the molten agar base at a temperature of 45 – 50 °C.
3.  

If the medium is not used on the day of preparation, it must be stored in a tightly closed container to prevent drying. It is important to note that in order to avoid the loss of stability of ready-made liquid media, it should not be stored for a long time. Agar media, also to avoid deterioration, should not be exposed to prolonged exposure to high temperatures (40-50 ° C).

When working, preference should be given to freshly prepared media. Nutrient media with such labile substances as beta-lactam antibiotics should be used within a few days after preparation to avoid loss of activity.

Cups from agar medium should be stored at + 2 … 8°С in tightly closed containers to prevent moisture loss. Liquid media in test tubes and vials should also be hermetically closed. Evaporation of moisture can lead to crystallization of some components of the environment.

The stability of prepared media is limited and varies considerably. If there are no special instructions, then the medium can be stored at 12 … 15 °C for several months. It is not recommended to store media at negative temperatures, as this will disrupt the gel structure.

Before sowing, the cups are carefully checked for the absence of contamination, unevenness of the agar surface, bubbles, color changes, hemolysis, and tears caused by drying of the medium. Test tubes and cups with such defects are thrown away.

1. Laboratory conditions: the microbiological laboratory is dangerous for the uninitiated and untrained, so the degree of risk is reduced for those who know about the possible dangers and the principles of safe behavior in the laboratory. The conditions under which microbial cultures are manipulated should always be taken into account. Most countries have adopted standards for such conditions, depending on the type of microorganisms used and the category of the microbiological laboratory. To work with the most dangerous microorganisms, it is suggested to implement a full set of safety measures. Failure to follow these instructions and regulations may constitute an offence.
2. Personnel actions: only qualified personnel trained in the handling of microbiological materials are allowed to work with infected material and inoculated nutrient media. All test material samples and microbial cultures should be handled properly and allowed to be discarded without autoclaving. The user must make sure that there is no microbial contamination of the equipment and apparatus, and if it is suspected, use disinfectants or autoclaving. Unnecessary microbial cultures in glassware (vials, test tubes, Petri dishes) should first of all be destroyed by autoclaving (approximately 30 min at 121 ° C).
3. Biological safety: it is necessary to understand that sowing microorganisms on nutrient media leads to a sharp increase in their number. At such high concentrations, any microorganism is potentially dangerous, so to prevent contamination, microbial cultures must be disposed of by appropriate safe methods.

All dry nutrient media are supplied in the form of a fine powder. These products are intended for bacteriological work in the laboratory, as indicated on the appropriate labels, and should not be used directly or indirectly by humans or animals. Most of the products are fine volatile powders, so inhalation should be avoided to avoid irritation of the upper respiratory tract. Avoid prolonged contact of the powder with damaged skin or excessive blooming of the powder. Any remaining powder should be washed off with plenty of cold water.

It is recommended to use individual protective masks to prevent the ingress of nutrient medium powder aerosols into the respiratory tract.

Each nutrient medium is accompanied by special instructions for preparation and use.

Danger symbols

Most nutrient media may contain toxic substances as components, so they must be handled as indicated in the “Safety Precautions” section of the instructions. If there are any toxic substances in the material, the label has the appropriate designations and phrases.

Sodium azide: Usually, when used in any environment, its concentration does not exceed 1%, which corresponds to low toxicity. However, some individuals are hypersensitive to this substance, so precautions should be taken to prevent inhaling or ingesting sodium azide powder. This substance has the property of reacting with many metals, especially copper and lead, with the formation of explosive compounds – metal azides. In this regard, it is recommended to strictly follow the provisions of local or national legislation on the disposal of sodium azide. To prevent long-term contact with metal gutters and sewer pipes, the remains of this powder should be washed off with a large amount of water. The same measures are necessary when using any biological fluid containing sodium azide as a preservative.

Lithium Chloride: This salt is a hazardous substance, so contact with any part of the body or inhalation of vapors should be avoided. If lithium chloride gets on the skin, it should be washed with plenty of water.

Cycloheximide: This is a highly toxic compound, so skin contact, aerosolization, and inhalation should be avoided.

Fuchsin basic: Is a potential carcinogen, so care should be taken to avoid inhalation of its powder or skin contact.

Sodium hydroselenite: This is a highly toxic compound with teratogenic and corrosive properties, so special care is required when handling it. If sodium hydroselenite gets on the skin, it should be washed with plenty of water.

First aid

In the event of accidents involving the careless handling of dangerous or toxic substances, the following first aid measures are recommended for victims.

Inhalation: Remove the victim from the area affected by the substance, warm him up and, if necessary, seek medical help.

Skin contact: Immediately remove all clothing contaminated with the substance and thoroughly wash the contaminated skin area with soap and water. If there are symptoms of poisoning after the above procedures, seek medical help.

Ingestion: Rinse mouth thoroughly with plenty of water. Immediately let the victim drink 2-3 glasses of water. If there are symptoms of poisoning after the above procedures, seek medical help.

Eye contact: Wash eyes thoroughly with plenty of water. If necessary, seek medical help.

Spilling (spilling) of material: In case of spilling or spilling of material, the following should be done:

1. A) with a large amount – wear protective overalls, gloves, glasses and a mask; collect the material in a container and close it carefully; disposal of material – in accordance with current legislation; remove the remains of the substance with a large amount of water;
2. B) with a small amount – wear protective gloves and remove the remains of the substance with a large amount of water.

BE CAREFUL WHEN USING DRY MEDIUMS

Some of the possible errors in the preparation of media

In order to obtain optimal results, it is important to store HiMedia products under appropriate conditions. Products with an expired expiration date should not be used as intended. Storage conditions and shelf life are indicated on the appropriate labels, containers and in the instructions-inserts. It is recommended to use products in ascending order of batch and batch numbers.

Attitude to light

It is advisable to store all prepared nutrients in the dark and always away from direct sunlight.

Attitude to temperature and humidity

Due to hygroscopicity, dry nutrient media are damaged in a humid atmosphere, so it is important not to leave containers with them open for a long time. High temperature storage or medium room conditions are not suitable for storing medium containers, especially those that are opened frequently.

Recommended storage time and temperature

Dry nutrient media (M)

Unopened dry nutrient media, when stored in optimal conditions, have a shelf life of 2-5 years.

Dried nutrient media manufactured by HiMedia are supplied in opaque, water-repellent plastic bottles with a screw-on cap that has an internal lid, so no additional capping is required. After taking the medium, the cap should be returned to its place and carefully bury the bottle with it.

Unopened containers of media should be stored below 25°C or below 8°C unless otherwise directed (see label).

When opening a vial or other container for the first time, the date of opening is written on it.

Selective additives (FD)

It is recommended to store at + 2 … 8 ° C, the shelf life is from 1 to 3 years.

Discs with antibiotics (SD, OD)

Stored at minus 20 °C, working batches – at +2…8 °C. The storage period is from 1 to 3 years.

Components of nutrient media (RM)

Unopened containers with media components should be stored at a temperature below 25 ° C, agar-agar – below 30 ° C, and horse serum – at a temperature below minus 20 ° C.

Materials for the differentiation of microbes

It is recommended to store at + 2 … 8 ° C, with the exception of factors V and X (at minus 10 ° C) and discs with carbohydrates (below 30 ° C); storage period – from 9 months to 2 years.

The creation of nutrient media for microorganisms requires a balanced assessment of both the individual ingredient and the interaction of all components with each other. Aware of this, HiMedia specialists apply appropriate control methods at various stages of production, which allows obtaining high-quality nutrient media. Their use ensures the necessary reproducibility of research results.

Quality control of individual components

The composition of dry nutrient media includes various components:

• Peptone (nitrogen source);
• Carbohydrates (source of carbon);
• Mineral substances (inorganic salts, trace elements);
• Selective substances;
• Vitamins;
• Dyes and pH indicators;
• Gel-forming component.

1. Peptones (meat, casein, soy, gelatin, yeast, etc.): Protein hydrolysis products, usually called peptones, are a mixture of polypeptides, oligopeptides, amino acids, organic sources of nitrogen, salts, and trace elements. The use of different peptones reflects the different needs of microorganisms in amino acids and peptides. The characteristics of peptone depend on the protein sources (casein, meat or soy) and the type of hydrolysis: acid or enzymatic (trypsin, pepsin or papain).

In order to control the produced peptones, in addition to its own complex of researches, the HiMedia company conducts an analysis according to the US Pharmacopoeia (section “Peptic Digest of Animal Tissue”) for compliance with the criterion “Pancreatic Digestion of Casein” (14).

Common tests for the analysis of peptones

1. Degree of digestion;
2. Loss at 100 ° C;
3. Nitrogen content;
4. Content of a-amino nitrogen;
5. Residue after burning (ash);
6. Absence of nitrites;
7. Salt content (for example, NaCl);
8. Phosphate content;
9. Microelements;
10. Fermentable carbohydrates;
11. Lipids;
12. Vitamins;
13. Compatibility with other ingredients at 121 ° C for 15 minutes;
14. Evaluation by microbiologists: a) by detection of specific metabolites (indole, acetylmethylcarbinol, hydrogen sulfide); b) according to growth properties (the degree of turbidity is assessed on liquid media, on dense media – the characteristics of colonies of microorganisms according to the method of Miles and Misra (12)).
15. Carbohydrates.

Some microorganisms can utilize a wide range of carbohydrates, others are more picky or utilize only one carbohydrate. The latter can be identified by their fermentation or oxidation of carbohydrates added to the environment. In the diagnostic practice of bacteriology, a large number of nutrient media are used, the composition of which includes specific carbohydrates and indicators for the detection of special enzymatic reactions (2, 4, 6, 7, 8, 9, 10, 13).

Carbohydrates used in nutrient media are checked for authenticity and absence of impurities. Control is carried out in the following way. Prepare liquid and dense nutrient media with one known carbohydrate – standard and tested. Then the ability of carbohydrates to support the growth of microorganisms is tested. On dense media, after sowing and incubation, compare the characteristics of the colonies on the medium with the standard carbohydrate and with the tested one (by size, color, shape, and reaction of the indicators).

3. Mineral substances and other components:

Some mineral salts are necessary for the growth and metabolism of all living cells. Most bacteria require the presence of sodium, potassium, magnesium, manganese, bi- or trivalent iron in the form of salts (chlorides, phosphates, sulfates) in the nutrient medium.

4. Selective substances (salts of bile acids and bovine bile)

Substances obtained from bile are introduced into nutrient media for the differentiation of bacteria adapted and unadapted to living conditions in the intestine.

Selective media with an increased concentration of bile and its derivatives are prepared for the differentiation of bacteria by tolerance to bile.

Chemical analysis of bile salts is performed as described in the National Formulary for sodium deoxycholate, sodium cholate, and sodium taurocholate (B.P. 1949 14 – U.S.P.).

In the nutrient medium, these substances should not affect the initial color of the indicator color and its subsequent changes during the growth of microorganisms.

At the same time, the medium should not foam or give sediment during storage. Next, a functional test of bile is carried out, using standard bile salts as a control. A test is also conducted to isolate the reference strain of E. coli on media with the tested and standard bile salts.

Dense nutrient media with various bile salts are tested by the surface drop method according to Miles and Misra (12).

Growth characteristics of different enterobacteria are recorded for each environment.

5. Dyes and pH indicators

Dyes added to nutrient media are used as selective substances, as well as as indicators of pH or redox potential. Their inhibitory activity may depend on the interaction with other components of the environment. Thus, the activity of diamond green in relation to E. coli varies significantly in solutions of different peptones. Bile salts interact with diamond green, as a result of which the toxicity of the paint decreases, and this should be taken into account when introducing such dyes into an environment with bile salts.

Aniline dyes are more toxic in a state of full oxidation, and when they are sterilized in the presence of peptone (as part of the broth), their partial recovery is possible. Also, when using agar with a high content of mineral substances, incompatibility of the components of the medium is often observed. In this regard, it is necessary that the dyes used in the preparation of nutrient media are controlled according to “H.J. Conn’s Biological Stains”, 9th edition, published by the Williams & Wilkins Company, Baltimore. Aniline dyes are more toxic in a state of full oxidation, and when they are sterilized in the presence of peptone (as part of the broth), their partial recovery is possible. Also, when using agar with a high content of mineral substances, incompatibility of the components of the medium is often observed. In this regard, it is necessary that the dyes used in the preparation of nutrient media are controlled according to “H.J. Conn’s Biological Stains”, 9th edition, published by the Williams & Wilkins Company, Baltimore.

6. Gelling agent.

The main purpose of agar-agar in a nutrient medium is to form a gel of a certain density. The transparency of the agar nutrient medium is also an important characteristic. Ideally, the molten agar should be crystal clear, with no evidence of turbidity or sediment. The cause of turbidity is the incompatibility of mineral substances or the smallest particles getting into the environment (as a result of ineffective filtration). Another characteristic of agar is the diffusion parameters of various chemical compounds in the agar gel. This property of agar media is used for microbiological evaluation of vitamins and antibiotics. Diffusion occurs from a reservoir located at a certain point on the agar. At the same time, a microbial growth retardation zone is formed around the tank, the diameter of which is directly proportional to the activity of the antibiotic or a growth zone proportional to the activity of the vitamin.

Agar-agar for bacteriological pu

rposes must meet the following chemical parameters:

In addition, a test is conducted for the presence of toxic substances and growth inhibitors of microorganisms in the agar. To do this, fast-growing microbes are sown on the tested nutrient medium and medium with standard agar-agar, followed by a comparison of the growth rate, size, shape, pigmentation of the colonies, etc.

7. Vitamins

Analysis of vitamins and their precursors for chemical purity and other tests is carried out in accordance with the US Pharmacopoeia (14) and the US Food Chemical Codex (7).

In addition to chemical analysis, the activity is confirmed by microbiological methods according to A.O.A.C. (3).

Quality control during production:

Samples are prepared from tested and approved components and analyzed in parallel from a predetermined reference batch of the finished medium.

A large series of batches is approved for production after evaluation of all characteristics, including color, clarity, pH, solubility, gelation, ingredient compatibility, and cultural characteristics of the sample. In the production process, according to the principles of GMP (good manufacturing practice), each stage is given the closest attention (14).

At the intermediate stages of production, samples are taken and delivered to the control laboratory for microbiological evaluation according to its own criteria developed by the HiMedia company. In most production processes, such conditions as temperature, humidity, etc. are constantly monitored. Production is carried out on stainless steel or glass equipment, which prevents contamination of products with toxic metals.

Finally, the finished products are checked in a dry form according to a number of physical parameters to ensure the homogeneity of the environment: appearance, color, smell, moisture, solubility, transparency, pH, gelation temperature (for agar media). In order to avoid the instability of the characteristics from batch to batch in the obtained environments in comparison with the ready-to-use reference environment, cultural properties are studied. The medium must pass all the tests planned by the HiMedia quality control laboratory: growth properties, differentiation, biochemical parameters, detection of growth when seeding a minimal inoculum, selectivity, etc. After passing these tests, the products are sent to the retail network.

Research of stability during warehouse storage of final products:

Samples of fully tested products and protocols compiled during their production are stored for future comparative studies.

After 6 months, a comparative study of batch samples using reference media is carried out to confirm stability.

Safety rules

Appeal and use

Read the label before opening the container.

Make sure that the product you need is in front of you.

Be aware of potential hazards when handling reagents and use appropriate protective clothing and equipment.

The container should be opened carefully, in a well-ventilated area.

Be careful when obtaining and using dangerous reagents, use methods that reduce the risk of poisoning by inhalation, ingestion, skin, eyes, or clothing.

Avoid using contaminated tools and equipment.

After use, carefully close the containers.

When working with reagents, eating, drinking or smoking is not allowed.

Wash contaminated hands and clothing thoroughly.

If the reagent is spilled or spilled, follow the instructions for safety measures.

If the reagent affects the body, immediately seek medical help, giving the victim first aid, and observe him until the doctor arrives.

Storage:

1. In necessary cases, the storage temperature is indicated on the label. All other products are stored at a temperature below 30 ° C.
2. Materials should be stored in a dry, well-ventilated place without sudden temperature fluctuations, away from open flames.
3. Do not allow unauthorized persons to access the reagents.
4. Hazardous substances must be stored separately.
5. Periodically, it is necessary to carry out an audit and remove damaged materials from the warehouse.
6. Do not allow smoking in places where flammable reagents are stored.
7. Be careful with containers where there may be residues of dangerous substances.

1. American Public Health Association – Standard Methods for Examination of Dairy Products, 14th edition, 1978
2. American Public Health Association – Compendium of Methods for Microbiological Examination of Food, 1976
3. Association of Official Analytical Chemists, 14th edition, 1984
4. Clinical Bacteriology by E. Joan Stokes & G.L. Ridgway, 5th edition, 1980
5. Cruickshank R., Duguid, J.P., Marmion , B.P. & Swain, R.H.A., 1975, Medical Microbiology, 12th Edition, – Churchill Livingstone, Edinburg, London, New York.
6. FDA Bacteriological Analytical Manual, U.S.A., 5th edition, 1979.
7. Food Chemical Codex, 3rd edition, 1981.
8. Laboratory Procedures in Clinical Microbiology, 2nd edition, 1985 by John A. Washington Published by Springer-Verlag, New York.
9. Manual of Clinical Microbiology, 4th edition by American Society for Microbiology, Washington D.C., 1985.
10. Media for isolation-cultivation-identification & Maintenance of Medical Bacteria. Vol. I, by Jean F.MacFaddin, Williams & Wilkins. Baltimore, 1985.
11. Methods in Carbohydrate Chemistry Vol I, Academic Press, U.S.A.
12. Miles A.A. & Misra S.S. (1938) “The Examination of Bactericidal Power of Blood” J. Hyg. Camb 38, 732-748
13. Standard Methods for Examination of Water and Wastewater 1985, 16th edition, APHA, AWWA, WPCF.
14. U.S. Pharmacopeia XXI National Formulary XVI, 1985.