GENERAL AND FOOD MICROBIOLOGY PART I - L. V. Krasnikova - 2016

APPENDIX

Class="center">Culture MEDIA AND Reagents used in microbiological practice

The basis of most culture media is meat infusion; for Yeasts, it is brewer's wort; for nutrient-demanding lactic acid Bacteria, it is hydrolysed milk; and for some others, it includes Liver broth, Hottinger digest, etc.

1. Media for the cultivation of mesophilic and thermophilic aerobic and facultatively anaerobic microorganisms

1.1. Nutrient broth (NB). To prepare NB, meat infusion is prepared first. For this purpose, beef free of bones, fat, and tendons is cut into small pieces or passed through a meat grinder. The minced meat is mixed with cold Water at a ratio of 1 dm3 per 500 g of meat and left in a refrigerator for 12–18 hours. The next day, the mixture is slowly heated over a fire and boiled for 15–20 minutes with occasional stirring. The cooled mass is filtered through a cotton-gauze filter, and water is added to restore the initial volume. To the resulting meat infusion, 1% peptone and 0.5% sodium chloride are added, and the required pH is adjusted by adding a saturated sodium bicarbonate solution or a 10% sodium hydroxide solution. It is sterilised at a Temperature of 121±1 °C for 15–20 minutes.

1.2. Nutrient Agar (NA). Before sterilisation, 20 g of agar is added to 1 dm³ of NB and heated over low heat until the agar is completely dissolved. The hot NA is filtered through a cotton-gauze filter, the pH is adjusted within the range of 7.2–7.4, the medium is dispensed into flasks or test tubes, and it is sterilised at a temperature of 121±1 °C for 20 minutes.

NA can be prepared from dry nutrient agar manufactured by the microbiological industry. To do this, 50 g of dry nutrient agar powder is transferred into a closed container with 1 dm³ of cold tap water and boiled over low heat until the agar is completely dissolved for 1–2 minutes with stirring. The pH of the medium is adjusted to 7.0–7.2, the solution is filtered through cotton wool, and it is sterilised at a temperature of 121±1 °C for 20 minutes.

1.3. Nutrient broth (agar) with glucose and Yeast extract. Before sterilisation, 10 g of glucose and 2 g of yeast extract (or 10 cm³ of its solution) are added to 1 dm³ of nutrient broth (agar). The pH of the medium is adjusted to 7.0–7.2, and it is sterilised at a temperature of 121±1 °C for 20 minutes.

1.4. Yeast extract. 100 g of compressed baker's yeast is crushed and mixed with 500 cm³ of tap water that has been boiled and cooled to 60 °C. The mixture is placed in an incubator at a temperature of 58–60 °C for 72 hours, with daily shaking 1–2 times. During this process, autolysis of the yeast Cells occurs, cellular Enzymes hydrolyse complex substances, and the yeast mass liquefies. The yeast extract is filtered, dispensed into small portions, and sterilised at 121±1 °C for 15 minutes. The ready-to-use extract is best stored in a refrigerator.

2. Media for the cultivation of yeasts and moulds

To detect and enumerate yeasts and moulds in food products, culture media are used whose selectivity is achieved by a low pH value and the Introduction of broad-spectrum Antibiotics (tetracycline, levomycetin, neomycin, etc.) to suppress the accompanying bacterial flora.

2.1. Sabouraud medium. 18 g of agar is added to 1 dm³ of distilled water and left for 30 minutes to swell, after which 40 g of glucose and 10 g of peptone are added. The mixture is warmed over low heat until the agar is completely melted. The molten medium is filtered through a cotton-gauze filter, and the pH is adjusted so that it is 6.5–6.6 after sterilisation. It is dispensed into flasks and sterilised for 15 minutes at a temperature of 121±1 °C.

Sabouraud medium is used as a base for preparing media with antibiotics. The Use of the medium without antibiotics is permitted when analysing canned products for commercial sterility.

2.2. Unhopped brewer's wort. Ready-to-use unhopped brewer's wort is diluted with water until the mass fraction of dry matter is 7–8%, dispensed into flasks or test tubes, and sterilised for 20 minutes at a temperature of 116±1 °C.

2.3. Wort agar. 20 g of agar is added to 1 dm³ of diluted brewer's wort. The medium is melted in a water bath and filtered through a cotton-gauze filter. The filtrate is dispensed into flasks or test tubes and sterilised for 15 minutes at a temperature of 116 °C.

3. Media for the cultivation of coliform bacteria

3.1. Endo medium. 100 cm³ of nutrient agar (pH 7.4) is melted and cooled to a temperature of 70 °C. 1 g of chemically pure lactose, previously dissolved in a sterile test tube with a small amount of distilled boiled water, is added.

In separate test tubes, prepare 2–3 cm³ of a saturated alcoholic solution of basic fuchsin and 10 cm³ of a 10% aqueous solution of sodium sulphite.

Measure 1 cm³ of fuchsin into a sterile test tube and add the sodium sulphite solution until the fuchsin is decolorised (pale pink colour). The prepared mixture is poured into the molten agar, mixed thoroughly, and poured into Petri dishes. The hot pale pink agar becomes colourless upon solidification.

Endo medium is manufactured by the domestic microbiological industry in the form of a dry powder. The medium is prepared on the day of use According to the instructions on the label.

3.2. Eijkman glucose-peptone medium. 10 g of peptone and 5 g of sodium chloride are dissolved in 100 cm³ of water, the mixture is heated to a boil, filtered, and 10 g of glucose is added. The pH is adjusted to 7.4–7.6, the medium is dispensed into test tubes or bottles with Durham tubes, and it is sterilised at 0.05 MPa for 20 minutes.

3.3. Kessler medium. 10 g of peptone and 50 cm³ of fresh ox Bile are added to 1 dm³ of water. The mixture is boiled with stirring until the peptone is completely dissolved, filtered through cotton wool, 2.5 g of lactose is dissolved, the volume is adjusted to 1 dm³, and the reaction of the medium is adjusted (pH 7.4–7.6). Then, 2 cm³ of gentian violet is added, the medium is dispensed into test tubes or flasks with Durham tubes, and it is sterilised at a temperature of 121 °C for 15 minutes.

Kessler medium is manufactured by the domestic microbiological industry in the form of a dry powder. In laboratories, it is prepared according to the instructions indicated on the label.

3.4. Simmons citrate agar. The medium is designed to determine the ability of enterobacteria to utilise citrates.

Preparation. Dissolve the medium components in distilled water and boil for 2–3 min until the agar is completely melted. The pH of the medium should be 7.0–7.2. Dispense the medium into test tubes in 4 cm³ aliquots and sterilize at 112 °C for 20 min. Before use, melt the agar in the tubes using a water bath and allow them to solidify in a slanted position. The color of the prepared medium is olive-green.

Inoculation and Structure/127.html">Interpretation of Results. Apply a minimal amount of the inoculum, free from the original growth medium, onto Simmons citrate agar. Leave a control tube uninoculated. Incubate the cultures at 37 °C. Record the results every 24 h for 5 days. Microorganisms capable of utilizing citrate grow on Simmons medium and change its color from green to blue (positive reaction), whereas microorganisms incapable of utilizing citrate do not grow on this medium and do not alter its color (negative reaction).

3.5. MacConkey agar. This medium is used for the identification of bacteria of the family Enterobacteriaceae. Bile, bile salts, and crystal violet present in the medium inhibit the growth of concomitant microflora.

Preparation. Dissolve the ingredients in water by boiling for 1 min with continuous stirring. Adjust the pH of the medium to 7.1±0.2. Dispense the medium into flasks, plug them with cotton-gauze stoppers, cover the tops with paper caps, and sterilize at 121 °C for 15 min.

Inoculation and interpretation of results. Streak the inoculum to obtain isolated colonies. Incubate the Petri dishes at 37 °C for 24 h.

Escherichia coli colonies are red; Klebsiella mobilis colonies are pink; enterococci colonies are small, round, and red; staphylococci colonies are light pink and opaque; Pseudomonas aeruginosa colonies are greenish-brown and fluorescent.

4. Media for Anaerobic Cultivation

4.1. Wilson-Blair medium. This medium is used to determine the sulfite-reducing capacity of mesophilic anaerobic microorganisms, particularly for detecting Clostridium perfringens in food products.

Preparation. Prepare the ferric ammonium alum and sodium sulfite solutions using sterile distilled water. Sterilize the sodium sulfite solution with flowing steam for 1 h. To 10 ml of molten meat-peptone agar with glucose cooled to 80 °C, add 10 ml of sodium sulfite solution and 1 ml of ferric ammonium alum solution. Adjust the pH of the medium to 7.5–7.8. Pour the medium into Petri dishes.

Inoculation and interpretation of results. Inoculate with infected material or streak from an enrichment medium using the double-layer method. Incubate for 18–24 h under anaerobic conditions at 37 °C. Sulfite-reducing clostridia form black colonies due to The production of iron sulfide.

5. Stains and Reagents for Gram Staining

5.1. Phenolic gentian violet solution. Gentian violet – 1 g; 96% ethanol – 10 cm³; crystalline phenol – 2 g;

distilled water – up to 100 cm³.

5.2. Lugol's solution. Potassium iodide – 2 g; crystalline iodine – 1 g; distilled water – up to 300 cm³. First, prepare a concentrated solution of potassium iodide in 5 cm of water, dissolve the iodine in it, and then add water up to 300 cm³.

5.3. Pfeiffer's fuchsin. Aqueous solution of Ziehl's fuchsin: 1 cm³ of Ziehl's carbol fuchsin and 9 cm³ of distilled water.

5.4. Ziehl's fuchsin. To prepare Ziehl's fuchsin, dissolve 1 g of basic fuchsin in 10 cm³ of 96% ethanol, then add 5 g of phenol dissolved in water and a few drops of glycerol. Bring the volume of the solution to 100 cm³ with distilled water and let it stand for several days. Filter before use.