FUNDAMENTALS OF MICROBIOLOGY - E. Yu. Tyumentseva - 2015

TOPIC 2. WORKING WITH MICROORGANISMS

Microorganisms inhabited the Earth 3-4 billion years ago, long before The Emergence of Higher Plants and animals. Microbes represent the most numerous and diverse group of living organisms. They are extremely widespread in nature and are the only forms of living matter that inhabit any and all diverse substrates (habitats), including more highly organized animal and plant organisms.

Because microorganisms are extremely small, their size is measured in micrometers: 1 millimeter (mm) = 1000 micrometers (µm) = 1,000,000 nanometers (nm) = 10,000,000 angstroms (Å).

Microorganisms shaped the atmosphere, drive the global cycles of matter and energy, facilitate The breakdown of Organic compounds and Protein Synthesis, and contribute to soil fertility, The formation of petroleum and coal, rock weathering, and many other natural phenomena.

Microorganisms are essential for key Industrial processes, such as baking, winemaking and brewing, as well as the Production of organic acids, Enzymes, dietary Proteins, Hormones, Antibiotics, and other pharmaceutical products.

When studying, identifying, and classifying microorganisms, the following genotypic and phenotypic characteristics are most frequently examined:

1. Morphological - shape, size, specific spatial arrangement, and Structure.

2. Staining (Tinctorial) - affinity for various Dyes (staining patterns), most notably the Gram stain. Based on this property, all microorganisms are divided into Gram-positive and Gram-negative.

3. Cultural - the growth characteristics of a microorganism on nutrient media.

4. Biochemical - The ability to ferment various substrates (CARBOHYDRATES, proteins, Amino Acids, etc.), and to produce diverse biochemical compounds through metabolic activity driven by specific enzyme systems and metabolic pathways.

5. Antigenic - determined primarily by the chemical Composition and Structure of The Cell wall, as well as the presence of flagella or capsules; recognized by the host's ability to produce Antibodies and Other immune responses, and detected via immunological reactions.

6. Physiological - modes of carbon Nutrition (autotrophs, heterotrophs), nitrogen nutrition (aminoautotrophs, aminoheterotrophs), and other Nutritional types, as well as Respiration types (aerobes, microaerophiles, facultative anaerobes, obligate anaerobes).

7. Motility and types of movement.

8. Spore-forming capacity and spore characteristics.

9. Susceptibility to Bacteriophages, and phage typing.

10. Chemical composition of cell walls - principal sugars and amino acids, lipid and fatty acid profiles.

11. Protein spectrum (polypeptide profile).

12. Susceptibility to antibiotics and other pharmacological agents.

13. Genotypic (utilization of molecular systematics Methods).

2.1. CULTIVATION OF MICROORGANISMS

2.1.1. Terminology

The propagation of microorganisms on nutrient media is termed cultivation (from Latin cultus - cultivation), and the resulting growth is referred to as a culture. When grown in a liquid medium, cultures form a suspension, sediment, or pellicle; on solid media, they form colonies. A culture may be a pure culture, containing the progeny of a single cell type, or an enrichment culture, consisting predominantly of Cells of one microbial species.

The Introduction of microbial cells or a test sample (such as soil or Water) into a sterile nutrient medium to obtain a pure or enrichment culture is called inoculation. Transferring previously grown cells from one medium to a fresh sterile medium is termed subculturing or passage (from Latin passus - step).

Microorganisms are typically grown at a controlled, constant Temperature inside incubators (wooden or metal cabinets) or temperature-controlled rooms. In both cases, a constant temperature is maintained using thermoregulators.

Cultivation at a specific temperature is called incubation (from Lat. incubatio – rearing, hatching of chicks).

Microorganisms are grown in glassware such as test tubes, flasks, or Petri dishes. To prepare new glassware, it is cleaned of alkali by boiling in a solution containing K₂Сr₂О₇ (6 %) and concentrated H₂SO₄ (6 %).

In test tubes, microorganisms are cultivated in both liquid and solid media. Liquid medium for aerobic cultures usually fills 1/3 of the tube, while for anaerobic cultures it fills 2/3. If a solid medium in test tubes is intended for subsequent microorganism cultivation, it is poured to 1/3–1/4 of the tube volume during preparation for sterilization.

After sterilization, test tubes with the medium not yet solidified are laid out on a flat table surface in a tilted position (at a slight angle) to obtain a slanted Agar surface. These are the so-called slants or slanted media.

A solid medium that has solidified while the test tube is in a vertical position is called a stab or butt. Nutrient medium butts, occupying 1/3–1/2 of the tube volume, are used for stab inoculation. Nutrient medium butts occupying 2/3 of the tube volume are used after sterilization for pouring into sterile Petri dishes intended for microbiological inoculation.

During work, test tubes with media and cultures are placed in racks; test tubes with media prepared for sterilization are placed in wire baskets or perforated metal buckets; test tubes with cultures during incubation or storage are placed in cardboard boxes.

When cultivating microorganisms in flasks, only liquid nutrient media are used. For aerobic microorganisms, the medium is poured in a thin layer (e.g., 30 ml in a 100 ml Erlenmeyer flask), whereas for anaerobic microorganisms, the flask is filled to 2/3 of its volume.

In Petri dishes, microorganisms are cultivated exclusively on solid media. The height of this glassware is about 1.5 cm and the diameter is 8–10 cm, with the diameter of the upper dish (which serves as a lid) being slightly larger than that of the lower one.

Special bacteriological needles, loops, and spatulas are used for working with microorganisms. They are made of platinum wire secured in special metal holders or sealed into Glass rods. The thickness of needles and loops should not exceed 0.5 mm, while the thickness of a spatula can be 1.5 mm or more.

When inoculating and subculturing microorganism cultures from colonies grown on solid media, needles or spatulas are used. The latter are also used for harvesting microorganism cells from colonies that grow into the substrate. Microorganism Suspensions are taken with a loop.

When preparing microorganism smears, glass slides are held suspended using Cornet forceps or special forceps-holders. It is advisable to dry the smears on the upper tier of a Koch metal drying table. They are conveniently washed on crossbar fixtures or so-called slide holders—parallel glass rods connected by rubber tubes (rod length 20–30 cm, tube length 15–20 cm). The rods are set over porcelain dishes or baths.

2.1.2. Inoculation Technique

Inoculation (or subculturing) is always performed near a burner. When transferring microorganism cells from one test tube to another, both tubes (one with the culture, the other with the sterile nutrient medium) are held in the left hand. One test tube is clamped between the index and middle fingers (the first test tube). Its lower end rests freely on the thumb (on its left side). The second test tube is clamped between the middle and ring fingers. It should lie parallel to the first one. Its lower end is located on the right side of the thumb. The thumb, positioned between the test tubes, should remain in a natural, relaxed state, gently holding the tubes parallel to each other.

When taking a smear, the test tubes must be kept in an inclined position to ensure the sterility of the culture. If the test tubes are positioned vertically, extraneous microorganism cells from the air may enter.

The bacteriological needle (loop) is thoroughly flamed in the burner while holding it in the right hand in a vertical position. Using the little finger of the right hand, the cotton plug is removed from the second test tube and clamped between the little finger and the palm. The plug of the first test tube is clamped between the ring and middle fingers of the right hand. The needle is flamed briefly again and inserted into the culture tube. The platinum needle cools down very rapidly. By touching it lightly to a microorganism colony, a small amount of microbial mass is gathered and transferred to the second test tube.

When inoculating a solid slanted medium with a culture-carrying needle, a straight or wavy line is drawn across its surface with a light motion, without damaging the medium—this is known as streak inoculation.

When inoculating into a nutrient medium butt, the needle is inserted into the bulk of its central part—this is known as stab inoculation. When inoculating into a liquid medium (or from a liquid medium), the test tubes are tilted only slightly to avoid wetting the plugs and the rims of the tubes.

Before using the plugs to close the test tubes, they are flamed. It is more convenient to close the first test tube first, and then the second one.

2.2. METHODS FOR PREPARING Microorganism Smears

Several methods for preparing microorganism smears are distinguished. This topic covers the technique of culture collection for smear preparation, METHODS FOR STUDYING microorganisms (such as the hanging-drop and press-preparation methods), the preparation of fixed smears, and types of stains.

2.2.1. Technique of Culture Collection for Smear Preparation

The test tube containing the culture is held in the left hand in a near-horizontal position close to the burner. Before collecting the culture, the cotton plug is removed from the test tube with the right hand, clamping it between the little finger and the palm, and the rim of the test tube is flamed over the burner. The needle is held in the right hand using the thumb, index, and middle fingers. Using the bacteriological needle sterilized in the flame, a small amount of microbial mass is taken from the test tube.

If the culture is taken from a liquid medium, the test tube should not be tilted too much to avoid wetting its rim and plug. It is preferable to use a loop for collecting the culture. After collection, the rim of the test tube and the plug are flamed, and the tube is closed.

2.2.2. Investigation of Living Microorganism Cells by Hanging-Drop and Press-Preparation Methods

In both cases, staining of the object is performed using vital dyes—vital staining. Methylene blue and neutral red at concentrations ranging from 0.001 to 0.0001 % can serve as vital dyes.

Both methods are used to study the motility of microbial cells, observe reproduction, spore formation and germination, determine how microorganisms respond to chemical compounds and physical environmental factors, examine cell dimensions and arrangement patterns, and identify intracellular storage compounds.

The specimens are examined under the Microscope with a slightly dimmed field of view; the condenser is lowered slightly, and the light intensity is adjusted using a concave mirror. Observations begin with a low magnification using an 8x objective. Once the edge of the droplet is located, either a 40x or an immersion objective (90x) is brought into place. Sharper and more detailed results can be achieved using dark-field or Phase-contrast Microscopy.

When using the hanging-drop method, a drop of tap water is placed on a clean glass slide. A microbial culture is introduced into the drop and mixed thoroughly with the water. A coverslip is then placed over the drop, taking care to prevent the formation of air bubbles underneath. The coverslip is gently pressed against the slide using a glass rod, and excess water is removed by touching filter paper to the edges of the coverslip. When examining the prepared specimen under an oil immersion objective, a drop of cedar oil is applied directly onto the coverslip.

This method is well-suited for investigating bacterial motility as well as observing larger objects such as microscopic Fungi and Yeasts. It is also applied when studying intracellular reserve Materials.

For long-term observations of microbial cells, the hanging-drop method is employed. This technique requires a specialized concave microscope slide featuring a depression in the center. Using an inoculation needle, a sparse suspension of microorganisms—either grown in a liquid nutrient medium or specifically prepared for the task—is placed onto a sterile coverslip.

2.2.3. Fixed Microbial Specimens

In microbiology, fixed preparations are frequently prepared and examined under a microscope in a stained state. Fixation refers to the Treatment of a living specimen that rapidly halts its life processes while preserving its fine ultrastructure. As a result of fixation, cells adhere firmly to the glass slide and take up stains more effectively. Fixation is also mandatory when working with pathogenic microorganisms for safety reasons.

Preparation of a smear. A drop of tap water is placed onto a clean, degreased glass slide. Slides are typically degreased using a 1:1 mixture of ethyl alcohol and diethyl ether. These Procedures must be performed well away from open burners. Using a sterilized bacteriological loop or needle, a small amount of microbial biomass is collected from a culture tube and mixed into the water drop. The drop is then carefully spread across an area of approximately 4 cm² on the glass using the loop.

If the original suspension is too dense, it must be diluted with water beforehand. To do this, a loopful of the dense suspension is transferred into a drop of water on a separate glass slide. Once the suspension reaches the optimal density, it is smeared in a thin layer across the slide. The smear is then air-dried at room temperature or by gentle heating, holding the slide high above a burner flame. Rapid or excessive heating during drying is not recommended, as it causes protein coagulation, which distorts Cell Structure and Morphology. Once fully dried, the preparation is fixed.

Fixation of the smear. Fixation is performed over a burner flame when studying cell morphology, or using chemical agents when investigating intracellular structure. In the first case, the underside of the slide is passed slowly through the burner flame three to four times. In the second case, chemical fixatives such as chromium compounds, formalin, osmium tetroxide, or acetone are utilized.

A common fixation technique involves treating the specimen with 96% alcohol or an equal-volume mixture of ethyl alcohol and ether (Nikiforov's fluid). For this, slides are immersed in the fixing solution for 10–30 minutes.

Staining the specimen. During staining, the slide is placed on a specimen holder. Several drops of stain are applied directly onto the smear. Depending on the type of stain and the objectives of the study, the staining duration ranges from 1 to 5 minutes, occasionally extending to 30 minutes or more. Upon completion, the slide is rinsed with water, excess moisture is removed with filter paper, and the preparation is air-dried before microscopic examination.

There are both simple and differential staining methods available.

Simple staining utilizes a single dye, such as methylene blue, fuchsin, or crystal violet in alkaline or carbolic solutions. In this technique, the entire microbial cell is uniformly stained.

Differential staining highlights specific cellular structures using multiple distinct dyes. Examples include the Gram stain and various spore-staining techniques.

In addition to the Gram stain, complex differential staining techniques include:

1. Ziehl-Neelsen acid-fast staining:

- the heat-fixed smear is stained for 3–5 minutes with Ziehl's carbol fuchsin solution (or a fuchsin-stained strip of paper), gently heating until vapors appear, without letting the stain boil;

- the slide is allowed to cool, the paper strip is removed, excess stain is drained off, and the slide is rinsed with water;

- the stained preparation is decolorized using a 5% aqueous solution of H₂SO₄ (sulfuric acid) for 3–5 seconds, or with 96% ethyl alcohol containing 3% Hydrochloric acid by volume, by repeatedly dipping the slide into a Coplin jar containing the solution;

- following decolorization, residual acid is rinsed away with water;

- the specimen is counterstained with Löffler's methylene blue for 3–5 minutes, rinsed with water, air-dried, and examined under the microscope.

Staining results: when stained using the Ziehl-Neelsen method, acid-fast Bacteria appear bright red due to the fuchsin stain.

2. Romanowsky-Giemsa staining.

The Romanowsky-Giemsa stain consists of a mixture of azure, eosin, and methylene blue. Before use, 10 drops of the stain are added to 10 ml of distilled water. The prepared staining solution is applied to the fixed smear and left for 1 hour. Afterward, the stain is drained, the slide is rinsed with water, and air-dried. The Romanowsky-Giemsa stain colors microorganisms in shades of violet-red.

To stain microorganisms, acidic and basic dyes are used. The former react with basic substances, while the latter react with acidic substances. Since proteins contain both basic (NH₂) and acidic (-COOH) radicals, cellular structures readily take up both types of dyes.

Among basic dyes, the most commonly used in microbiology include: green — Janus green, methylene green, malachite green; blue — Victoria blue, methylene blue; purple — gentian violet, crystal violet, methylene purple; red — neutral red, safranin, fuchsin, hematoxylin; brown — vesuvin, chrysoidine; black — induline.

Acidic dyes may include the following: black — nigrosine; red and pink — acid fuchsin, erythrosine; yellow — Congo red, picric acid, fluorescein.

Basic dyes stain objects more intensely in an alkaline environment, whereas acidic dyes do so in an acidic environment. To differentiate between solutions of acidic and basic dyes, filter paper strips are immersed in them. These strips carry a negative electrical charge. In the case of a basic staining solution, its cations—which are responsible for the coloring properties—are fixed by the negative charge of the paper, meaning that only water will travel up the paper via capillarity (forming a colorless band). If the solution contains an acidic dye, its anions will move up the paper and stain it.

Staining living microorganism cells (vital staining) is a highly labor-intensive process and is therefore rarely used in microbiology. Microbial cells are fixed not only for safety reasons (if the cultures are pathogenic) or to anchor them to the glass slide, but also to enable them to absorb the dye more effectively. It is believed that fixation increases the number of free amino and carboxyl groups, thereby enhancing the cellular affinity for the dye.

Dyes can be categorized into positive and negative.

Positive dyes directly stain microorganism cells and other objects. The majority of dyes used in microbiology are positive. They stain cells at room temperature within 30–60 seconds.

Negative dyes stain the Background surrounding the microbial cells. As a result, the cells appear as silhouettes against a colored background.

Certain microorganisms (such as spirochetes) and specific structures (such as extracellular slime) that are poorly visualized with positive dyes are clearly revealed by negative dyes. Spores do not take up stain without appropriate treatment; consequently, when bacterial cells are stained with positive dyes, spores appear as light-refracting inclusions within the vegetative cells.

Laboratory Procedure

Objective: to study inoculation techniques, methods for preparing microbial smears, and smear fixation; to learn staining methods and dye Classification. To develop skills in preparing fixed bacterial smears and master the technique of simple staining for bacteria.

Materials, Reagents, and equipment: microscope; test tubes; flasks; Petri dishes; bacteriological needles; loops; spatulas; stands; glass microscope slides and coverslips; gentian violet; Lugol's solution; toothpicks; spirit lamp; 96% ethyl alcohol; filter paper; fuchsin; wash bottle.

Experiment No. 1. Study of Spirochetes

a) Preparation of a dental plaque smear.

1. Collect a small amount of dental plaque using the sharp end of a toothpick.

2. Spread it on a microscope slide to about the size of a coin.

3. Fix the smear by passing the slide through a burner flame three times.

4. Stain the smear using the Gram method.

5. Rinse with water.

6. Dry with filter paper and in air.

7. Examine under the microscope.

b) Gram staining of the smear.

1. Flood the smear with a small amount of gentian violet; staining time is 2 min.

2. Pour off the excess dye into a waste tray, apply several drops of Lugol's solution to the smear using a pipette, and leave for 1 minute.

3. Pour several drops of alcohol onto the smear, decolorizing until the purple dye runs off in streams, but for no longer than 30 seconds.

4. Thoroughly rinse the smear with water.

5. Counterstain the smear with diluted fuchsin for 2 minutes.

c) Microscopy of the specimen.

1. Set up the illumination: the condenser must be raised to its highest position, and adjustments should be made using the 8x low-power objective—a uniformly bright, white field of view is required.

2. Place the specimen on the microscope stage.

3. Using the coarse adjustment knob, lower the objective until it is approximately 0.5 cm away from the specimen.

4. Looking through the eyepiece, bring the specimen into focus by turning the coarse adjustment knob counterclockwise (toward yourself).

5. Perform fine focusing using the fine adjustment knob.

6. Switch the nosepiece to high magnification (40x objective) and adjust the focus using only the fine adjustment knob.

7. After examining the specimen, switch the nosepiece back to the 8x (low) magnification objective, and only then remove the slide from the stage.

Oral spirochetes are extremely thin, nearly Hair-like, and short (possessing only 2–3 turns).

Preparation and Analysis of Research Results

In their reports, students should provide a brief Summary of the theoretical material. The microscopic structures observed must be sketched, followed by a Conclusion regarding the morphology of dental plaque. Below the drawings, indicate the magnification used and label the name of the object studied.

As a result of this practical work, students will master inoculation techniques, methods for preparing microbial smears and fixing them, staining methods and classification of dyes, and the Gram-Staining procedure for microorganisms, as well as acquire practical microscopy skills.

Control Questions

1. Define the following terms: “cultivation,” “pure culture,” “enrichment culture,” “passaging,” and “incubation.”

2. How is sampling of a pure microbial culture performed?

3. What is the procedure for sampling a culture to prepare a specimen?

4. List the techniques used for microbial inoculation. How are they performed?

5. Outline the step-by-step procedure for microscopy.

6. How is smear fixation carried out?

7. What is THE PRINCIPLE OF the hanging-drop method?

8. What is the principle of the press-mount (crushed drop) method?

9. List the main steps in preparing a fixed, stained smear.

10. What methods are used for staining microorganisms?

11. How is Gram staining performed?

12. How are the Ziehl-Neelsen and Romanowsky-Giemsa stains performed?

13. List the main stains.

14. Name the acidic stains.