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

7. STUDY OF BACTERIAL MORPHOLOGY

Objective of the work: to study bacterial Morphology.

Determining the species of Bacteria (identification) relies on a combination of characteristics, including morphological ones (from Greek morphus – form), which encompass Cell shape and size, Gram-staining properties, motility (presence or absence of flagella), and The ability to form endospores and capsules.

7.1. Bacterial Shapes

Based on their external appearance, three primary bacterial forms are distinguished: spherical (cocci), rod-shaped (cylindrical), and spiral/convoluted (Fig. 7.1). Pathogenic agents of infectious diseases are found among spiral forms, making them key subjects in medical microbiology.

Spherical bacteria are typically spherical in shape, though some resemble candle flames, lancets, beans, or coffee beans. Depending on how Cells arrange themselves following division, cocci are classified into:

micrococci (from Greek micros – small) – single cells or cells arranged in no particular order;

diplococci (from Greek diplos – double) – occurring in pairs because the cells remain attached after division (e.g., Lactococcus);

streptococci (from Greek streptos – twisted, braided) – round or elongated cells forming chains due to Cell Division occurring in a single plane while maintaining connection between the cells;

tetrads (from Greek tetra – four) – groups consisting of four cocci;

sarcinae (from Latin sarcia – to bind) – packet-like clusters of cocci formed by division in three mutually perpendicular planes, consisting of 8 or more cells;

staphylococci (from Greek staphyle – grape bunch) – cell clusters resembling bunches of grapes resulting from division across multiple planes;

gonococci (the causative agents of Gonorrhea) – shaped like coffee beans joined in pairs with their concave sides facing each other;

pneumococci and meningococci – resembling a candle flame in shape and arranged in pairs with their broad bases facing inward.

Class="center">Fig. 7.1. Main bacterial shapes (Vorobyov et al., 1994): 1 – staphylococci; 2 – streptococci; 3 – sarcina; 4 – gonococci; 5 – pneumococci; 6 – pneumococcal capsule; 7 – Corynebacterium diphtheriae; 8 – clostridia; 9 – bacilli; 10 – vibrios; 11 – spirilla; 12 – treponema; 13 – borrelia; 14 – leptospira; 15 – actinomycetes; 16 – flagellar arrangement: a – monotrichous, b – lophotrichous, c – amphitrichous, d – peritrichous

Rod-shaped bacteria possess a cylindrical form varying in length and diameter, and also differ in the shape of their cell ends and mutual cellular arrangement. The ends of the rods may be truncated (Bacillus anthracis), tapered (Fusobacterium), rounded (Escherichia coli), or swollen at the extremities to resemble a club (Corynebacterium diphtheriae).

Rods may occur singly, in pairs, chains, palisades, or rosettes. Among rod-shaped bacteria, there are endospore-forming species—bacilli (from Latin bacillus – staff) and clostridia (from Greek closter – spindle)—as well as non-spore-forming bacteria (Pseudomonas, Escherichia coli, Salmonella, Proteus, etc.).

Pathogens of diphtheria belonging to the genus Corynebacterium are characterized by the presence of polyphosphate granules at the ends of the rods. Rods capable of forming branching structures are classified as mycobacteria (Mycobacterium tuberculosis, actinomycetes, bifidobacteria).

Spiral bacteria. Convoluted and spiral forms include:

vibrios (from Latin vibrio – to bend) – slightly Curved Rods resembling a comma in shape (Vibrio cholerae);

campylobacters – cells exhibiting bends similar to a seagull's wing;

spirilla – mildly spiral forms with cells having 3–5 turns;

spirochetes – highly coiled, slender, long, flexible cells featuring numerous turns. Pathogenic species affecting humans are found among spirochetes, specifically Representatives of the genera Leptospira, Treponema, and Borrelia.

7.2. Gram Staining of Bacteria

Based on this characteristic, all bacteria are divided into two groups:

✵ Gram-positive (stain with Gram's method);

✵ Gram-negative (do not stain with Gram's method).

The method is based on differences in the chemical Composition and Structure of the Introduction/37.html">Bacterial Cell wall.

The Essence of the method lies in the differences in the Chemical Composition and STRUCTURE OF THE bacterial cell wall.

Gram Staining Technique

1. Place three drops of Water in three spots on a degreased Microscope slide and prepare three thin smears of different bacterial species: use controls at the edges (with a known Gram reaction) and the test culture smear in the center.

2. Allow the smears to air dry and heat-fix them over a spirit lamp flame.

3. Stain the smears with gentian violet for 1 min (place a strip of filter paper soaked in the stain onto the slide and moisten it with water).

4. Remove the paper strip and, without rinsing the slide with water, apply Lugol's iodine solution. Leave it for 1 min (until the smear turns completely black).

5. Without washing with water, treat the slide with 96% alcohol for 15-20 s, continuously rocking the microscope slide. It is very crucial to strictly adhere to the decolorization time, as exceeding it will also decolorize Gram-positive bacteria.

6. Rinse the slide with water, place a strip of filter paper soaked in Pfeiffer's fuchsin on it, moisten with water, and stain for 1 min.

7. Remove the paper strip, rinse the slide with water, and blot dry with filter paper.

8. Apply cedar oil to the slide and examine under an immersion objective.

Following this Procedure, Gram-positive bacteria stain purple, while Gram-negative bacteria stain red.

Gram-positive bacteria include micrococci, streptococci, staphylococci, bacilli, clostridia, and lactic acid bacteria. Gram-negative bacteria include Escherichia coli, salmonellae, brucellae, the causative agents of dysentery and cholera, acetic acid bacteria, pseudomonads, etc.

7.3. Detection of Bacterial Motility

Many species of bacteria are capable of independent movement due to the presence of flagella. The arrangement and number of flagella serve as a diagnostic feature in identifying bacterial species.

To detect bacterial motility, "hanging drop" or "crushed drop" preparations are made using young (24-hour) broth cultures of bacteria. Under microscopic observation, the active movement of individual cells in various directions and at different speeds is clearly visible in the field of view.

7.4. Bacterial Spore Staining

Due to their structural features, bacterial spores exhibit high resistance to various unfavorable environmental factors. During standard staining with methylene blue, spores do not take up the stain and appear as colorless round, oval, or elliptical bodies that refract light strongly. This is because the spore coat is very dense, has a high lipid content, and free water is absent within the spore itself. Spore staining Methods are based on The Use of mordants (usually weak acids that loosen the spore coat) coupled with subsequent staining of the smear using heat. The stained spore protoplast retains the dye more firmly than the Cell Cytoplasm; therefore, unlike the cytoplasm, it is not decolorized during subsequent acid Treatment. The decolorized cytoplasm is then counterstained with a contrasting dye.

Ziehl-Neelsen Method

1. Prepare a fixed bacterial smear in the usual manner.

2. Apply a 5% chromic acid solution to the smear and let it stand for 5-10 min.

3. Wash the acid off with water, place a strip of filter paper on the slide, and saturate it thoroughly with carbol fuchsin. Gently heat the slide over a spirit burner until vapors appear (do not bring to a boil), then remove it from the flame and add another portion of the stain. Continue this procedure for 5–7 min. It is important to ensure that the stain does not evaporate and the paper does not dry out.

4. After the slide cools, remove the paper, rinse the slide with water, and treat it with a 1% solution of hydrochloric or sulfuric acid for 15–30 s. When preparing smears of spore-forming bacilli such as B. subtilis, B. mycoides, or B. mesentericus, it is recommended to decolorize The Cell cytoplasm for 16–18 s.

5. Stain the smear with methylene blue for 1–2 min, then wash off the stain, blot the slide dry with filter paper, and examine it using an oil immersion objective.

With this staining method, endospores appear red, while vegetative cells appear blue.

Peshkov's Method

1. Flood the prepared fixed smear with Loeffler's methylene blue and, holding the slide over a flame, boil it for 15–20 s, preventing the stain from drying out.

2. Rinse the smear with water and counterstain with a 0.5% neutral red solution for 30 s.

3. Rinse the slide with water, blot dry, and examine under an oil immersion objective.

Using this staining technique, spores stain light blue or blue, whereas the cell cytoplasm stains pink.

To identify bacteria, it is essential to determine the type of sporulation (bacillar, clostridial, or plectridial), THE POSITION OF the endospore within the cell (polar, central, or subterminal/eccentric), and the shape of free spores (spherical, oval, or cylindrical). For this purpose, 2- to 3-day-old cultures of spore-forming bacteria are used.

7.5. Detection of Bacterial Capsules

Some microorganisms form capsules, particularly when grown on carbohydrate-rich media. The presence of a capsule serves as a diagnostic feature in bacterial identification. Standard bacterial staining techniques leave capsules unstained; therefore, special staining methods are employed for their visualization.

Gins-Burri Capsule Stain. Dilute liquid India ink with distilled water in a 1:10 ratio. Mix a drop of bacterial suspension on a microscope slide with a drop of the diluted ink, blend thoroughly, and spread it into a thin film across the slide. Air-dry the smear and examine it under an oil immersion objective. Against the smoky dark Background of the slide, bacterial cells appear surrounded by distinct unstained capsules.

Review Questions

1. What METHODS FOR STUDYING bacterial morphology are you familiar with?

2. What is the underlying principle and technique of the Gram stain?

3. What are the Specific features of staining bacterial spores?

4. What methods can be used to determine bacterial motility?

5. Which method is used to detect bacterial capsules?