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

2. ORGANIZATION AND EQUIPMENT OF A MICROBIOLOGY LABORATORY

Objective: to familiarize students with the equipment of a microbiology laboratory, as well as the Methods and Means of sterilizing culture media, glassware, and equipment.

A microbiology laboratory should include the following rooms:

✵ laboratory rooms for conducting research;

✵ a Glass-enclosed hood/booth for performing operations that require a high degree of sterility;

✵ a washing room equipped for cleaning glassware;

✵ a preparation room adapted for preparing culture media, preparing glassware for sterilization, and making Reagents;

✵ a sterilization area equipped with autoclaves. All laboratory rooms must have good natural and artificial lighting. Table surfaces and floors should be covered with easy-to-clean Materials (plastic, linoleum); in the sterile booth, autoclave room, and washing room, walls and floors are tiled. To sterilize the air in the laboratory rooms, bactericidal lamps (such as BUV-15, BUV-30, etc.) are installed.

Equipment: autoclaves, incubators, hot-air ovens, microscopes, refrigerators, technical balances, apparatus for microbiological air analysis of rooms, colony counters, anaerostats for cultivating anaerobic Bacteria, pH meters.

Glassware and supplies: bacteriological loops and needles, Drigalski spatulas, scissors, forceps, racks, Laboratory glassware (Petri dishes, test tubes, pipettes—most commonly 1.0 and 0.1 cm³, Microscope slides and coverslips, dropping bottles for stains and solutions), cotton wool, gauze, filter paper.

2.1. Methods and Means of Sterilization Used in Microbiological Practice

Sterilization (from Latin sterilis - barren) in microbiological practice and the food industry refers to the destruction of all vegetative microbial Cells and their spores in materials. Sterilization is one of the most crucial and essential Procedures in microbiological practice.

The following Methods of sterilization exist:

Thermal sterilization

✵ direct flaming (incineration);

✵ boiling;

✵ dry heat;

✵ saturated steam under pressure;

Cold sterilization

✵ filtration;

✵ irradiation;

Disinfection.

Thermal sterilization

Flaming - sterilization by heating small items to high temperatures in the flame of an alcohol burner or Bunsen burner (glass slides, bacteriological loops, needles, forceps, lancets, etc.). The necks of flasks and test tubes are also flamed during culture transfer and the pouring of culture media.

Boiling is used to sterilize metal instruments, needles, and rubber tubing. The instruments are boiled in special metal sterilizers with lids for 30–40 minutes. However, even very prolonged boiling does not ensure complete sterility, as the spores of certain anaerobic clostridia can survive boiling for up to 3 hours.

Dry-heat sterilization. Glassware (pipettes, test tubes, flasks, Petri dishes, etc.) is kept in a hot-air oven at 160 to 180 °C for 1.0–1.5 hours. This Treatment destroys both vegetative cells and microbial spores.

Before sterilization, glassware is washed and dried. Test tubes and flasks are plugged with cotton-gauze plugs. Test tubes are wrapped in paper bundles of 10–20. Paper caps are placed over the flasks to protect the necks from dust. Cotton plugs are inserted into the mouthpiece ends of pipettes. The pipettes are then placed in special glass or metal cylinders or wrapped in paper. During work, pipettes should be removed from the cylinder only by their upper end containing the plug. Petri dishes are wrapped in paper (individually or in sets of 2–3). The glassware prepared for sterilization is loaded into the drying oven without overcrowding to ensure uniform heating. Upon completion, the oven is turned off but should not be opened until the Temperature drops to 100–70 °C to prevent the glassware from cracking. To maintain sterility, the glassware is unwrapped immediately before use.

Steam sterilization under pressure is one of the most common and effective methods. It is based on the principle that steam generated by boiling Water accumulates in a confined space, thereby increasing the pressure. As the pressure rises, the temperature of the steam increases correspondingly (Table 2.1).

Class="center">Table 2.1. Correlation between pressure gauge readings and saturated steam temperature

Sterilization is carried out in special hermetically sealed double-walled apparatuses known as autoclaves. The operating principle of an autoclave is based on the increase in the boiling point of water as pressure rises. Water inside the autoclave is heated electrically to generate steam. The Main Components of an autoclave include the outer casing, the water-steam chamber, the sterilization chamber, and a lid with a rubber gasket. Additional accessories supplied with the autoclave include: a pressure gauge with a siphon tube and a three-way valve, a water gauge glass tube to monitor the water level in the water-steam chamber, a safety valve to prevent excessive pressure buildup in the autoclave, and air and exhaust Valves to remove air at the beginning of sterilization and to drain condensate from the sterilization chamber.

Autoclaves are used to sterilize culture media, physiological saline solutions, rubber items, and glassware with rubber stoppers. The sterilization regime (temperature and duration of treatment) is determined by the composition and pH of the nutrient medium. Media containing sugars, milk, or Vitamins are sterilized at 0,05 MPa for 15–20 minutes, whereas meat-peptone media are sterilized at 0,10 MPa for 20–30 minutes. The sterilization time is calculated from the moment the required pressure is reached.

At low pH values, certain Components of the nutrient medium may undergo Hydrolysis during sterilization. To prevent this, solutions of specific components (such as Amino Acids and vitamins) are sterilized separately and added to the medium after sterilization.

Streaming steam sterilization (fractional sterilization) is a method used to decontaminate culture media that alter their composition when exposed to temperatures above 100 °C. The Essence of fractional sterilization is that the medium is heated at 100 °C for 15–30 minutes on three consecutive days. The initial heating kills vegetative cells, while some spores survive and subsequently germinate. The vegetative forms arising from these heat-resistant spores are then destroyed during the subsequent heating cycles. Fractional sterilization is performed in a Koch steam sterilizer or in an autoclave with the exhaust valve open.

Tyndallization is a fractional sterilization Procedure for materials that are easily damaged by high temperatures (sera, vitamins, certain Antibiotics). Sterilization is accomplished by heating the material at 60 to 65 °C for 60 minutes on 5–6 consecutive days, or at 70 to 80 °C for 3 days.

Cold sterilization

Filtration through bacterial filters. Microfiltration using fine-pored filters is employed when elevated temperatures would drastically alter the quality of the materials being sterilized. Furthermore, filtration sterilization is utilized to purify Bacterial toxins and other metabolic products of microorganisms.

There are two MAIN TYPES OF filters: depth filters and membrane filters.

Depth filters consist of fibrous materials. Particles are retained within them through adsorption and mechanical entrapment within the filter matrix. Bacterial filters are manufactured from various materials and feature different pore diameters, which are specified on the packaging. Filter sterilization commonly employs Seitz asbestos pad filters, Chamberland candles made of kaolin mixed with quartz sand, and Berkefeld candles composed of diatomaceous earth and asbestos.

Membrane filters possess a continuous polymeric Structure, typically made of nitrocellulose, and particle retention is strictly determined by pore size. Membrane filters are designated by numbers from 1 to 5 depending on their pore diameter (350–1200 nm).

Sterilization by filtration is performed under a vacuum using a vacuum pump or water aspirator. Before operation, the filters are secured in a specialized holder connected to a Bunsen flask. The filtration assembly is autoclaved for 30–40 minutes at 0,15 MPa. Membrane and asbestos filters are intended for single use. Chamberland and Berkefeld candles, however, are washed after use by backflushing with distilled water and processed for reuse.

Radiation sterilization. Ultraviolet irradiation is used to eliminate microorganisms in indoor air and On the surface of food products prior to packaging. It is carried out using germicidal lamps of various capacities (wavelength 253–265 nm). Ionizing radiation is employed for the sterilization of certain packaging materials used in the food industry.

Disinfection

The destruction of microorganisms using chemical agents is termed disinfection (derived from the Latin infektia—infection, and the French negative prefix des-). Chemicals are applied to eradicate pathogenic microorganisms from environmental objects—workplaces, rooms, workwear, hands, Processing equipment, and utensils.

Substances used for disinfection must meet several key requirements:

✵ they must be readily soluble in water;

✵ they should exhibit rapid bactericidal action;

✵ they must not exert toxic effects on humans and animals;

✵ they should not damage the items being disinfected.

Disinfectants are classified into several groups:

1) chlorine-containing compounds (bleach, sodium hypochlorite, chloramine, pantocid, chlordezinsulfochlorantine, etc.);

2) iodine- and bromine-based compounds (iodopyrine, dibromantine);

3) oxidizing agents (hydrogen peroxide, potassium permanganate, etc.);

4) phenols and their derivatives (phenol, lysol, creolin, hexachlorophene);

5) heavy metal salts (sodium merthiolate, corrosive sublimate). Acids and their salts (boric, salicylic), alkalis, alcohols (70% ethanol solution), and aldehydes (formaldehyde) also exhibit antimicrobial activity.

Antibacterial soaps are also commercially available, such as phenolic, tar, and "Hygiene" soaps containing 3–5% hexachlorophene.

Review Questions

1. What methods of sterilization do you know?

2. Which sterilization methods are classified as thermal?

3. How should laboratory glassware be prepared for sterilization?

4. What factors determine the sterilization regimen?

5. In what cases are "cold" sterilization methods used?

6. What requirements are imposed on disinfectants?

7. What substances are used for disinfection?