7-5 BACTERIOLOGY

LEARNING OBJECTIVE: Recall bacteria classifications, common bacteria, and procedural steps for making smears, Gram staining, and reading and reporting smears.

Bacteriology is the study of bacteria. Of primary interest to medical technologist is medical bacteriology, which deals with the bacteria that cause disease in man.

Bacteria are prokaryotic microorganisms of the kingdom Protista. They reproduce asexually by transverse binary fission in which the cell divides into two new cells. Bacteria are found almost everywhere, and the human body harbors vast numbers. Many bacteria are beneficial and essential to human life; only a few are harmful to man.

BACTERIA CLASSIFICATION

Since there are thousands of types of bacteria, a method of classification is essential. Bacteria are classified according to their respective

Disease-Producing Ability

The disease-producing ability of bacteria is referred to as either pathogenic or nonpathogenic. Pathogens are bacteria that cause diseases, and nonpathogens are harmless bacteria. Bacteria that are essential to our body are, in their proper environment, called common or normal flora. For example, alpha streptococcus in the throat is common flora, but when it is found elsewhere (such as in the blood stream, possibly as a result of tooth extraction), it may cause diseases such as septicemia and endocarditis.

Growth Requirements

The four growth requirements for bacteria are

TEMPERATURE REQUIREMENTS.— Temperature requirements are divided into the following three categories.

OXYGEN REQUIREMENTS.—The amount of oxygen needed for an organism to grow or reproduce varies with the type of organism. Aerobes are organisms that reproduce in the presence of oxygen. Obligate aerobes are organisms that grow only in the presence of free oxygen. Anaerobes are organisms that do not reproduce in the presence of oxygen, and obligate anaerobes are organisms that grow only in the absence of free oxygen and are killed if exposed to free oxygen. Facultative organisms are organisms that grow in the presence of free oxygen and in an oxygen-free atmosphere. Microaerophilic organisms are organisms that grow only in low amounts of free oxygen.

NUTRITION REQUIREMENTS.—Nutrition requirements for the various types of bacteria depends on what their particular environment provides. Autotrophic bacteria are self-nourishing, and heterotrophic bacteria are not self-sustaining.

MOISTURE REQUIREMENTS.—Moisture is indispensable for bacterial growth.

Morphologic Characteristics

The structural (or morphologic) characteristics of bacteria are based on three distinct shapes or categories:

Three special structures, present on some bacteria, aid in the classification process of bacteria. The special structures are the capsule, the spore, and the flagellum. The capsule is a gummy, gelatinous, or mucoid structure surrounding certain bacteria. The spore is an inactive, resting, and resistant form produced within the organism, usually as a result of unfavorable environmental conditions. The third and final special structure is the flagellum, a hairlike structure that provides motility.

Colonial Morphology

A colony is a cohesive mass composed of many millions of bacterial cells, growing on or in a medium (such as blood agar, a gel enriched with blood that is used in the preparation of solid culture media for microorganisms) as a result of the multiplication and division of a single cell. The size, color, shape, edge, topography, consistency, and odor of the colony vary with each organism.

Toxins Produced

Generally, toxins produced are waste products of metabolism in a bacterial cell. Some bacteria produce toxins that attack red blood cells in a culture medium such as blood agar. Examples of toxins produced by bacteria are listed below:

Gram’s Stain Reaction

To differentiate and identify bacteria, you must make them visible by staining. The staining procedure, devised by Dr. Hans Christian Joachim Gram, stains microorganisms such as bacteria with crystal violet, treats them with 1:15 dilution of strong iodine solution, decolorizes them with ethanol or ethanol-acetone, and counterstains them with a contrasting dye, usually safranin. Microorganisms that retain the crystal violet stain (a dark blue-black color) are said to be gram-positive, and those that lose the crystal violet stain by decolorization but stain with counterstain (a deep pink or reddish color) are said to be gram-negative.

COMMON BACTERIA

Bacteria are named by genus and species. The first word (capitalized) indicates the genus; the second word (not capitalized) indicates the species, a subdivision of the genus. For example:

GENUS:

Neisseria

SPECIES:

gonorrhoeae

Table 7-3 will familiarize you with commonly encountered bacteria. This table lists the bacteria’s morphologic shape, Gram stain response, genus and species, and the type of infection it produces.

Table 7-3.—Common Bacteria

COMMON BACTERIA

Morphologic Shape Gram‑Positive or -Negative Genus & Species Type of Infection
Cocci Positive Streptococcus pneumoniae Pneumonia
Streptococcus pyogenes (Beta Streptococci Group A) Strep throat
Staphylococcus aureus Boils, furuncles, osteomyelitis,
pneumonia, septicemia, endocarditis, and impetigo
Negative Neisseria gonorrhoeae Gonorrhea
Neisseria meningitidis (meningococcus) Meningitis
Bacilli Positive Corynebacterium diphtheriae Diphtheria
Clostridium
(all are anaerobic and spore producers)
  • perfringens (welchii)
  • tetani
  • botulinum
Gas gangrene
Tetanus
Botulism
Negative Yersinia (Pasteurella) pestis Bubonic plague
Brucella abortus Brucellosis
Bordetella pertussis Whooping cough

BACTERIOLOGIC METHODS

There are a variety of methods used in the laboratory to identify bacteria. However, only a few of these bacteriologic methods can be performed in isolated duty locations or on board naval vessels. One of these methods is the smear. The smear permits healthcare personnel to examine specimens microscopically. Material requirements and the step-by-step procedures for making smears is covered in the following sections.

Smear

A smear is the procedure in which a specimen–a body fluid or a discharge–is spread across a glass slide for microscopic examination. To enhance the visualization of microorganisms on the smear, Gram staining (introduced earlier in this chapter) is used. Once the smear is stained, it is ready to be examined under the microscope. Normally, smears are examined by laboratory technicians who prepare reports of their findings.

MATERIALS REQUIRED FOR SMEAR.—To perform a smear, the following materials are required:

PROCEDURE FOR MAKING SMEARS.—To prepare smears for microscopic examination, follow these steps:

  1. Spread the specimen with a wood applicator stick across a slide that has been cleaned with alcohol or acetone and polished with lens paper. The smear should be thin and uniformly spread. If the smear is opaque, it is too thick and should be emulsified with a drop or two of saline.
  2. Label the smear and circle the material to be stained with a diamond point pen for easier identification and location of the material after staining.
  3. Let the smear air dry. Do not use forced heat drying; forced drying will distort bacterial cells and other materials.
  4. Hold the smear with forceps and fix the smear by passing it through a flame (smear side up) three or four times. Avoid overheating the smear; overheating will cause cellular wall destruction.
  5. Let the slide cool. Once the slide is cooled, it is ready to be stained.

Gram’s Stain

As previously explained, the most common staining procedure used in bacteriologic work is the Gram stain. This method yields valuable information and should be used on all smears that require staining. Gram’s stain is also used for examining cultures to determine purity and for identification purposes.

PRINCIPLE OF GRAM STAINING.—As touched on previously, the crystal violet stain, the primary stain, stains everything in the smear blue. The Gram’s iodine acts as a mordant, a substance that causes the crystal violet to penetrate and adhere to the gram-positive organisms. The acetone-alcohol mixture acts as the decolorizer that washes the stain away from everything in the smear except the gram-positive organisms. The safranin is the counter-stain that stains everything in the smear that has been decolorized: pus cells, mucus, and gram-negative organisms. The gram-negative organisms will stain a much deeper pink than the pus cells and mucus will stain even lighter pink than the pus cells.

MATERIALS REQUIRED FOR GRAM STAINING.—To Gram stain a smear, the following materials are required:

Gram stain kit, which consists of:

PROCEDURE FOR GRAM STAINING SMEARS.—After smears have been dried, heat-fixed, and cooled, proceed as follows:

  1. Place the slide on a staining rack. Then flood slide with primary stain (crystal violet). Let stand 1 minute.
  2. Remove the primary stain by gently washing with cold tap water.
  3. Flood the slide with mordant (iodine or stabilized iodine) and retain on slide for 1 minute.
  4. Remove mordant by gently washing with tap water.
  5. Tilt slide at a 45-degree angle and decolorize with the acetone-alcohol solution until the solvent that runs from the slide is colorless (30 to 60 seconds).
  6. Wash the slide gently in cold tap water.
  7. Flood the slide with counter-stain (safranin) and let stand for 30 to 60 seconds.
  8. Wash slide with cold tap water.
  9. Blot with blotting paper or paper towel or allow to air dry.
  10. Examine the smear under an oil immersion objective.

Reading and Reporting Smears

Place a drop of oil on the slide and, using the oil immersion objective of the microscope, read the

smear. All body discharges contain extraneous materials, such as pus cells and mucus. Of interest, however, are the types of bacteria that may be present. The stained smear reveals only two features: the morphology and the staining characteristics of the bacteria present. Positive identification requires cultures and further studies.

medical technologists should report only what they see. For example, “Smear shows numerous gram-negative bacilli.” If two or more types of bacteria are seen in a smear, the rule is to report them in order of predominance. For example: “Numerous gram-positive cocci in clusters", or "Few gram-negative bacilli”

Gram-positive organisms are easy to see because they stain a deep blue or blue-black. Gram-negative organisms stain a deep pink, but since the background material is also pink, minute and detailed inspection is necessary before reporting the results.

In the presence of gonorrhea, the smear will reveal large numbers of pus cells with varying numbers of intracellular and extracellular gram-negative, bean-shaped cocci in pairs. Such a finding could be considered diagnostic. It is important to point out that only a few of the thousands of pus cells on the slide may contain bacteria, and sometimes it requires considerable search to find one.