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

3. METHODS FOR PREPARING MICROBIAL SPECIMENS

Objective: to gain hands-on experience working with pure microbial cultures; to learn how to prepare fixed stained specimens, living (unstained) specimens, and impression smears of microorganisms.

In microbiological practice, the Morphology, biochemistry, and PHYSIOLOGY OF MICROORGANISMS are studied using pure cultures. A pure culture is defined as a population of microorganisms originating from a single Cell.

Working with pure cultures requires strict adherence to specific aseptic conditions to prevent contamination by extraneous microorganisms. All Procedures must be carried out within the sterile zone of a burning spirit lamp. Small items (Microscope slides, forceps, scissors, bacteriological loops, and needles) are sterilized by flaming (from the German flamme, meaning flame) in the spirit lamp's flame. Microorganisms grown on solid media are retrieved from the test tube using a bacteriological loop or needle. For broth cultures, a sterile pipette or loop is used.

3.1. Preparation of Fixed Stained Microbial Specimens

Purpose of fixation: to kill the microbial Cells and firmly attach them to The surface of the microscope slide so they are not washed away during staining and rinsing; to enhance cellular staining, as dead cells become more permeable to Dyes.

The Procedure for preparing a fixed stained specimen from a culture grown on an Agar slant in a test tube is as follows:

1. Light the spirit lamp.

2. Take a clean microscope slide and flame it by passing it through the spirit lamp flame several times.

3. Allow the slide to cool on the workbench and place a drop of Water from a dropper bottle onto it.

4. Hold the culture tube in your left hand between your thumb and index finger, tilting it so that the contents are clearly visible.

5. Take the bacteriological loop in your right hand, heat the wire to a red glow in the spirit lamp flame, and flame the portion of the holder adjacent to the wire.

6. Using the little finger and ring finger of your right hand, press the cotton plug against your palm, remove it from the tube, and hold it in this position without touching any surrounding objects.

Caution! Never place the plug on the workbench or other surfaces to avoid microbial contamination (from the Latin contaminatio, meaning bringing into contact).

7. Flame the rim of the opened test tube in the spirit lamp flame.

8. Insert the loop into the tube, cool it against the interior wall, and harvest a small amount of culture from the surface of the nutrient medium.

Caution! Be careful not to gouge or damage the nutrient agar with the loop.

9. Flame the rim of the tube again, re-insert the plug while passing the tube through the flame, and place the culture tube back into the rack.

10. Transfer the material from the loop into the drop of water on the microscope slide, gently mix it, and spread it into a thin, uniform film over the surface of the slide.

11. Thoroughly incinerate any remaining microbial cells on the loop in the burner flame, and return the loop to its stand.

12. Air-dry the microbial suspension in a stream of warm air above the spirit lamp flame, then fix it by passing the slide through the flame 3–4 times (never fix unfrozen or wet smears).

13. After cooling, place the slide on a staining rack, apply methylene blue, and let it stain for 2 minutes, timing it with an hourglass.

14. Rinse off the stain with water and gently blot the slide dry using filter paper by pressing it carefully against the Glass.

15. Apply a drop of cedar immersion oil directly onto the stained specimen. Place the slide on the microscope stage, use the coarse adjustment knob to immerse the oil immersion objective (90x) into the oil, and bring the microscopic field into view, fine-tuning the image clarity with the fine adjustment knob.

Attention! Do not use the fine adjustment knob when searching for microorganisms in a slide preparation.

3.2. Preparation of Live Microorganism Mounts

Live (unstained) mounts are used to study microorganisms in their active state to observe motility, spore germination, division patterns, etc. There are two primary techniques for preparing live mounts of microorganisms: the "hanging drop" and the "crushed drop" (wet mount) Methods.

Wet mount (crushed drop) preparation. Place a drop of water from a dropper bottle onto a clean microscope slide. Using an inoculation loop, transfer a small amount of the test culture into the drop and mix gently without spreading it widely across the slide. If the culture is grown in a liquid medium (as a microbial suspension), apply it directly to the slide using a pipette.

Cover the microbial suspension with a coverslip, taking care to avoid trapping air bubbles that could interfere with observation. Remove any excess liquid protruding from the edges of the coverslip by gently touching them with absorbent filter paper.

Place a drop of immersion oil directly onto the coverslip and examine the preparation using an oil immersion objective with the condenser lowered. Actively motile Bacteria will swim across the entire field of view, changing directions and performing rotational and circular movements.

Hanging drop preparation. This technique requires a specialized depression slide with a concave well in the center and a standard coverslip. Apply a thin layer of petroleum jelly around the rim of the well. Place a small drop of a broth culture (or suspension) of Microorganisms in the center of the coverslip. Invert the depression slide and lower it carefully over the coverslip so that the droplet on the coverslip aligns precisely with the center of the well.

Press the slide gently against the coverslip so they adhere securely, then lift the assembly and quickly flip it over so the coverslip faces upward. This creates a sealed chamber that prevents the drop from drying out for an extended period. In a properly prepared mount, the droplet should hang freely from the underside of the coverslip without touching the bottom or sides of the well. Place a drop of immersion oil on the coverslip and examine the preparation under a 90x objective in a dark-field configuration (using a narrowed aperture and a lowered condenser).

3.3. Preparation of Impression Smears (Replica Mounts)

Impression smears are used to study the natural spatial arrangement of microbial cells On the surface of a substrate or food product. Flame-sterilize a clean microscope slide and press it gently against the surface of the tested product (such as meat, fish, or cheese). Fix the impression smear by passing it briefly through a spirit lamp flame, then stain it with methylene blue or by the Gram stain method. Apply a drop of immersion oil and examine under the oil immersion objective.

3.4. Dyes and pH Indicators Used in Microbiological Practice

Without staining, microorganisms (except Fungi) remain virtually invisible under a Light Microscope due to their low optical contrast. In microbiological practice, staining is widely used to determine the shape, size, internal Structure, and spatial arrangement of microbes. Staining techniques for fixed smears are divided into simple and differential (complex) procedures. Simple staining utilizes basic and acidic aniline dyes. In basic dyes, the chromophore (the ion responsible for color) is a cation; in acidic dyes, it is an anion. Basic dyes bind much more intensely to the nuclear and acidic Components of the cell, whereas acidic dyes effectively stain cytoplasmic structures.

Basic dyes include: reds—neutral red, basic fuchsin, safranin, thionine, pyronin, hematoxylin;

✵ blues—methyl blue, Victoria blue;

✵ purples—crystal violet, gentian violet;

✵ greens—janus green, malachite green, methyl green;

✵ blacks—nigrosin (or similar variants).

Acidic dyes include:

✵ reds and pinks—acid fuchsin, eosin, erythrosine;

✵ yellows—congo red, picric acid, fluorescein;

✵ blacks—nigrosin.

Simple staining methods do not reveal fine cellular structures or differentiate the specific affinity of microbes for various dyes. Differential staining techniques are employed to achieve these goals. These include the Gram stain (see Section 7.2), the Ziehl-Neelsen acid-fast staining method, the detection of polyphosphate granules by Loeffler's method (see Section 6.3), bacterial spore staining via the Ziehl-Neelsen or Peshkov methods (see Section 7.4), capsule staining by the Gins-Burri method (see Section 7.5), and others.

pH indicators are Organic compounds that respond to shifts in the active acidity of a medium by changing color. Because microorganisms alter the acid-base balance of their growth environment during METABOLISM, indicators are used to monitor these metabolic shifts. Indicators that are colored in one pH range and colorless in another are termed single-color indicators, while those exhibiting different colors on either side of the transition interval are called two-color indicators.

Microbiologists most frequently utilize the two-color indicators listed in Table 3.1.

Class="center">Table 3.1. Two-color indicators

For example, when identifying microorganisms to determine their saccharolytic properties, the so-called Hiss's "differential series" of media is used. Hiss's media are prepared using 1% peptone water supplemented with 0.5–1.0% of a carbohydrate and Andrade's indicator (containing acid fuchsin) or bromothymol blue. The initial color of the medium (as well as in the absence of enzymatic activity) is straw-yellow with Andrade's indicator or blue-green with bromothymol

blue. Inoculated tubes containing Hiss's medium are incubated at the optimal Temperature for 24 hours. Microorganisms ferment CARBOHYDRATES with acid production, which alters the color of the medium: it turns pink-red with Andrade's indicator, and yellow with bromothymol blue.

Review Questions

1. What conditions must be observed when sampling a pure culture of microorganisms to prepare a slide?

2. What are the objectives of specimen fixation?

3. What methods are used to prepare viable (unstained) microbial preparations?

4. What simple and differential staining techniques for microbial preparations do you know?

5. What stains and dyes are used to color microorganisms?

6. What pH indicators are used in microbiological practice?