Microbiological Laboratory Techniques

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Microbiological Laboratory Techniques

LEARNING OBJECTIVES

After reading this chapter, the student will be able to:

• Describe the general concept of aseptic techniques used in laboratory preparation and analysis

• Explain and differentiate between sterilization, disinfection, and sanitization

• Describe the different types of culture media and their possible physical state

• Discuss inoculation, incubation, and isolation

• Describe the various fixation and staining techniques to identify microbes with the light microscope

• Describe the fixation and staining methods used in electron microscopy

• Classify the bacteria according to their shape for morphological identification

• Describe the different culture characteristics of microbes used for the purpose of identification

• Discuss the physiological, biochemical, and genetic characteristics used for microbial identification purposes

KEY TERMS

agar

antiseptics

aseptic technique

bactericidal

bacteriostatic

chemical defined media

complex media

contaminants

cultures

culture media

degermation

differential media

disinfection

enriched media

filamentous

fomite

fusiform

germicide

hemolysins

incubated

inoculation

inoculum

isolation

liquid media

nonsynthetic media

pellicle

peptone

pleomorphic

pour plate

sanitization

sediment

selective media

semisolid media

solid media

spread plate

sterile

sterilization

streak plate

synthetic media

the five “I’s”

turbid

WHY YOU NEED TO KNOW

HISTORY

To understand microbiology is to understand the laboratory; its basic equipment, how its equipment is used, the procedures carried out, and—importantly—the preparation of and meaning of test results.

In the 1600s, the newly created microscope yielded images of samples previously too small to be seen with the naked eye. Reports of interpretation of these early microbiological laboratory observations led to the realization that life could exist in a single cell, followed in the 1800s by the cell doctrine.

In addition, laboratory findings of Pasteur and Koch revealed pathogenic microorganisms to be causes and carriers of disease, from which evolved the germ theory of disease. In 1877, Pasteur and Joubert described microbial antagonism among bacteria and in 1899 the term “antibiotic” was coined. Present-day biotechnological methodologies have grown from laboratory findings that prompted the development of new technologies to drive further advances in microbiology as a scientific discipline.

IMPACT

Verification of the successes and failures of putative treatments for pathogenic diseases is performed under laboratory conditions in the search for new drug and antibiotic therapy. Moreover, results from the laboratory give basic understanding of microorganisms, their mechanisms of action, and their relationship with the environment.

The addition of clinical and laboratory experience permits a rational approach to the question of “what happens if?” and suggests pertinent experiments that can be carried out safely under carefully controlled laboratory conditions.

One of the beneficial outgrowths of microbiological investigations in the laboratory has been the need to use aseptic techniques for the growth and identification of specific microorganisms. Culture techniques were derived from the necessity to rapidly grow and accurately identify potential pathogens in order to treat individuals or take appropriate steps to prevent outbreaks of disease, epidemics, or pandemics.

FUTURE

The acknowledgment of threats to outbreaks of pathogenic disease has brought a public awareness to the importance of compliance with proven preventative procedures and therapies.

Aside from these ever-present battles against commonly confronted pathogens, another deadly issue has arisen—bacteriological warfare. Again, our most effective weapon against this potential disaster comes from the laboratory. Recently, the use of anthrax as a bioterrorism weapon was attempted in Washington, D.C. Through the prompt use of laboratory procedures, it was identified and safely disposed of, although weaponization of pathogens is and continues to be a bioterrorism threat.

Threats to human health via pathogenic organisms or via bioterrorism agents will be met by work done in laboratories. Laboratory work has been and continues to be the cornerstone of protection against disease, of microbiology, and of world health.

Aseptic Technique in Laboratory Preparation and Analysis

Medical and clinical laboratories test biological specimens to determine the health status of a patient and to identify the disease-causing pathogen for the purpose of appropriate treatment. Research laboratories include basic, clinical, and pharmaceutical laboratories, each dealing with a specific aspect of microbiological research.

Microorganisms are everywhere in the environment. In order to selectively identify specific microbes they must be grown in controlled laboratory environments. Beginning with pure sterile cultures, the key is to control the factors to which the cultures are subjected. In other words, when working with microbial cultures, it is necessary to ensure that organisms are selectively introduced into the culture and that other environmental organisms do not contaminate it. Aseptic technique is a procedure that is performed under sterile conditions, a method that prevents the introduction of unwanted organisms or contaminants into an environment. This process is characterized by strict adherence to details. The use of aseptic technique controls, limits, or prevents contamination by fomites. A fomite is any inanimate object or substance capable of transporting pathogens from one medium or individual to another. Aseptic technique is essential in the microbiology laboratory to prevent any contamination of laboratory personnel (see Chapter 5, Safety Issues), cultures, supplies, and equipment.

Air currents must be controlled by closing laboratory doors and windows to prevent microbes on surfaces from becoming airborne and entering the cultures. When handling cultures to prepare slides or to transfer organisms to another medium, the transfer loops and needles need to be sterilized by flame or incinerator before and after use (Figure 4.1). Furthermore, culture plates are held in a position that minimizes exposure of the medium to the environment. When removing lids/stoppers from test tubes, the lids should remain held in the hand and not placed on other surfaces such as countertops during the transfer of materials from one tube to another. Flaming is one of the physical methods of sterilization and must always be applied to the lips of test tubes and also of flasks whenever culture liquid is poured from one container to another, as in the case of pouring culture plates (Figure 4.2).

FIGURE 4.1 Sterilization of a loop.
One of the primary inoculation tools in the microbiology laboratory is the loop. Metal loops must be sterilized in the flame of a Bunsen burner by heating the wire until it glows. Presterilized, disposable plastic loops are also available for inoculation.

FIGURE 4.2 Aseptic technique and media containers.
Bacteria and fungi are everywhere in the environment, including the air. The mouth of a tube or bottle opening is a potential point of contamination and flaming of the opening in direct flame immediately after opening and before closing is an effective technique to prevent potential contamination on this exposed surface.

Sterilization and disinfection procedures are daily routines in microbiological laboratories. They are essential to ensure that cultures, containers, media, and equipment are handled in such a way that only the desired organism will grow and others will be eliminated or excluded.

Sterilization

Sterilization is the destruction or removal of all microorganisms, including bacteria and their endospores, viruses, fungi, and prions. This can be accomplished by physical methods such as heat, radiation, and filtration, or by chemical methods. The wide application of sterilization processes makes it necessary to impose strict control measures to validate the results. When using dry heat or moist heat sterilization, physical, chemical, or biological indicators can be used to validate the desired results (Table 4.1). The general resistance of microbes to methods of sterilization ranges from bacterial endospores, with the highest resistance to sterilization, to vegetative cells, with moderate to least resistance.

TABLE 4.1

Methods for Validating Dry or Moist Heat Sterilization

	Physical Methods	Chemical Methods	Biological Test Organism
Dry heat	Temperature-recording charts	Color change indicator	Bacillus subtilis var. niger
Moist heat	Temperature-recording charts	Color change indicator	Bacillus stearothermophilus

Physical and chemical methods of sterilization are applied in the microbiological laboratory to ensure that equipment and materials are free of microorganisms. Aseptic technique is the first and most important step in ensuring that manipulation of specimens during investigative procedures does not infect laboratory personnel or contaminate cultures or the laboratory environment (see Chapter 5, Safety Issues). Bacteria are found practically everywhere including fingertips and bench tops and therefore it is essential to minimize contact with these surfaces. Only sterile items are free of potentially contaminating microorganisms and once a sterile object comes in contact with a nonsterile surface, the object can no longer be considered sterile. The most commonly used instrument in the microbiology laboratory for the sterilization of media and glassware is the autoclave (Figure 4.3). A detailed discussion of the different types of physical and chemical methods of sterilization is provided in Chapter 19 (Physical and Chemical Methods of Control; for overviews see Tables 19.3 and 19.5).

FIGURE 4.3 Autoclave.
The autoclave is one of the primary tools for sterilization of equipment, containers, media, and biohazardous wastes. It is essentially a large pressure cooker that raises the temperature of steam to about 121° C under 15–20 psi pressure. The size and configuration of autoclaves vary but the basic operation is same.

Disinfection

Disinfectants are applied to inanimate surfaces, medical equipment, and other man-made objects whereas antiseptics are used to disinfect skin. The term disinfection refers to the use of a physical process or the use of a chemical agent to destroy vegetative microbes and viruses. This does not include bacterial endospores. The ideal disinfectant would result in complete sterilization without harming other forms of life. Unfortunately, ideal disinfectants as such do not exist and most of them only partially sterilize. In addition to the most resistant pathogens, endospores, other bacteria, and viruses are also highly resistant to many disinfectants.

Substances that kill bacteria are bactericidal and those that interfere with cell growth and reproduction are bacteriostatic. Disinfectants and antiseptics are bactericidal and bacteriostatic depending on the concentration applied. All disinfectants are by their nature potentially harmful, even toxic, to humans and animals. They should be handled with appropriate care to avoid harm to the handler or recipient. The type of disinfectant to be used depends on the surface or material to be disinfected. Specifics on the different types of disinfectants and their particular effectiveness are described in Chapter 19, with an overview in Table 19.5 (Chemicals Used in the Control of Microbes).

Sanitization

Several applications in everyday life and medicine do not require sterilization, disinfection, or antisepsis but need to reduce microorganisms in order to control possible infections or spoilage of substances. Sanitization achieves this by using any cleansing technique that mechanically removes microorganisms and other debris to reduce contamination to safe levels. Often the sanitizer used is a compound such as soap or detergent. Restaurants, dairies, breweries, and other food industries handle soiled utensils on a daily basis and must take appropriate measures to sanitize them for prevention of infection, spoilage, and contamination. This includes controlling microbes to a minimal level during preparation and processing.

Degermation is the process by which the numbers of microbes on the human skin are reduced by scrubbing, immersion in chemicals, or both. Some examples of degermation include the process of presurgical scrubbing of the hands with sterile brushes and germicidal soap before putting on sterile surgical gloves, the application of alcohol wipes to the skin, and the cleansing of a wound with germicidal soap and water.

Culture Techniques

Microbiologists use five basic procedures to examine and characterize microbes: Inoculation, Incubation, Isolation, Inspection (observation), and Identification—the five “I’s.” To culture a microorganism a small sample, the inoculum, is introduced into a culture medium usually with a platinum wire probe streaked across its surface. This process is called inoculation and the growth that appears on or in the medium is the culture. A culture can be pure—containing one type of organism, or mixed—containing two or more species.

Types of Culture Media

Nutritional requirements of particular microorganisms range from a few simple inorganic compounds to a complex list of specific inorganic and organic chemicals (Table 4.2). Access to carbon, the essential component required for molecular life, is obtained in different ways by microorganisms. Autotrophs acquire carbon from carbon dioxide in the atmosphere and heterotrophs obtain their carbon from organic compounds. This diversity is seen in the different types of media needed to ensure the growth of the organism for investigation. Media vary in nutrient content and consistency and can be classified according to their physical state, chemical composition, and functional type.

TABLE 4.2

Nutritional Requirements for Microorganisms*

Macronutrients	Growth Factors (Vitamins)	Micronutrients (Trace Elements)
Carbon	p-Aminobenzoic acid	Boron
Hydrogen	Folic acid	Chromium
Oxygen	Biotin	Cobalt
Nitrogen	Cobalamin (B₁₂)	Copper
Phosphorus	Lipoic acid	Iron
Sulfur	Nicotinic acid (niacin)	Manganese
Potassium	Pantothenic acid	Molybdenum
Magnesium	Riboflavin	Nickel
Sodium	Thiamine	Selenium
Calcium	Vitamin B₆	Tungsten
Iron	Vitamin K group	Vanadium
	Hydroxamates	Zinc

^*Not all microbes need all of the nutrients listed; therefore, for optimal growth environments media with specific nutrients are necessary for specific microorganisms.

Physical State of Media

Liquid media are water-based solutions that do not solidify at temperatures above freezing and flow freely in the containers when tilted. Most commonly, liquid media are supplied in tubes or bottles and are called broths, milks, or infusions. A common laboratory medium is nutrient broth, which contains beef extract and peptone (partially digested protein) dissolved in water. Methylene blue milk and litmus milk are opaque liquids prepared from skim milk powder and dyes. After inoculation, growth occurs throughout the container. Enriched broths are used to grow bacteria that are present in few numbers such as in small specimen samples obtained from patients.

Semisolid media contain a limited amount of a solidifying agent such as agar or gelatin, giving the medium a clotlike consistency. Semisolid media are often used to determine motility and growth patterns of bacteria.

Solid media are dispensed in Petri plates or slanted in tubes or bottles to provide firm and maximal surfaces for growing bacteria or fungi. By far the most widely used and effective of these media is agar, composed of a complex polysaccharide from the red alga Gelidium. Agar is solid at room temperature and liquefies at the boiling temperature of water. Once in liquid form it does not solidify until it cools to 42° C. It then can be inoculated and poured in liquid form at temperatures that will not harm the microbes or the handlers. Agar added to media simply gels them into a solid form. Any medium containing 1% to 5% agar usually has the agar in the name of the specific medium as, for example, nutrient agar, phenylethyl alcohol agar, blood agar, and others.

LIFE APPLICATION

Raising Bacteria: A Labor of Love

Most of the bacteria raised in laboratories for testing, identification, or experimentation grow on relatively simple media. Complex media such as tryptic soy broth/agar, nutrient broth/agar, brain heart infusion, and blood agar are useful for growing a wide range of bacteria. The conditions that most organisms are grown under vary from anaerobic to microaerophilic (5% oxygen) to capnophilic (“carbon dioxide loving”) to simply aerobic and in temperatures ranging from 4° C to about 60° C. These medium types and incubation conditions can be found and maintained in most microbiology/clinical laboratories. There are, however, some bacteria that are very picky eaters and require some extraordinary medium concoctions to raise them in the laboratory. The normal growth conditions of other bacteria, such as extremophiles, may be challenging to reproduce in the average laboratory. Some pathogenic bacteria such as those in the genera Spiroplasma and Mycoplasma need specialized media for culturing. Spiroplasma medium is a broth that contains more than 80 ingredients including various types of nutrients as well as antibiotics to suppress other possible competitors. Extremophiles, whose natural growth environment is at the vent holes at the bottom of the ocean, present some significant challenges for culturing. The extreme pressures and temperatures required as well as the unique nutritional requirements of these bacteria make their cultivation possible only in laboratories with sophisticated equipment to meet the organisms’ needs.

Chemical Classification of Media

Depending on their chemical content media can be classified as complex or nonsynthetic media and as chemically defined or synthetic media.

• Chemically defined media or synthetic media are media with a defined, exact chemical composition. They are prepared by means of an exact formula, adding precise amounts of inorganic and/or organic chemicals to distilled water. Some of these media contain minimal amounts of chemicals such as some salts and a source of carbon; others are special media containing a variety of precisely measured substances.

• Complex media or nonsynthetic media contain at least one component that cannot be chemically defined and thus the medium cannot be represented by an exact chemical formula. Complex media contain extracts from animals, plants, or yeast. They may include blood, serum, meat extracts, milk, yeast extracts, soybean digests, and peptone.

Functional Types of Media

General-purpose media are designed to grow a broad spectrum of microbes that do not have any special growth requirements. Other media are available for special growth conditions of selected organisms. These include enriched, selective, and differential media.

• Enriched media contain complex organic substances such as blood, serum, hemoglobin, or growth factors for the growth needs of specific species. An example is blood agar, made by adding sterile sheep, horse, or rabbit blood to a sterile agar base. It is widely used to grow certain streptococci and other pathogens. Another enriched medium is chocolate agar. Chocolate agar is enriched with heat-treated blood, which turns brown and gives the medium the color and thus its name.

• Selective media inhibit the growth of selected organisms while allowing the growth of others. These media are useful in isolating bacteria or fungi from specimens that contain several different organisms. For example, mannitol salt agar contains 7.5% NaCl, inhibitory to most human pathogens with the exception of the genus Staphylococcus, which thrives in mannitol salt agar and consequently its growth can be amplified in mixed samples.

• Differential media can grow several different organisms that show visible differences. These differences can be variations in colony size or color, a change in medium color, or the formation of gas bubbles and precipitates. Dyes can be used as differential agents because many of them are pH indicators that change color in response to acid or base production by a specific microbe. For example, MacConkey agar contains neutral red, which is a dye that is yellow when neutral and pink or red when acidic. Escherichia coli, a bacterium common to the intestinal tract, produces acid when it metabolizes the lactose in the medium and develops red or pink colonies. In contrast, Salmonella does not give off acid and therefore remains in a natural off-white color. A comparison of general, selective, and differential media is shown in Figure 4.4.