Culture System Lec. 3 PDF

Lec. 3 Microbial Diagnosis | Dr. Ahmad Hasan Culture System Growth and identification of the infecting agent in vitro is usually the most sensitive and specific means of diagnosis and is thus the method most commonly used. Theoretically, the presence of a single...

Lec. 3 Microbial Diagnosis | Dr. Ahmad Hasan Culture System Growth and identification of the infecting agent in vitro is usually the most sensitive and specific means of diagnosis and is thus the method most commonly used. Theoretically, the presence of a single live organism in the specimen can yield a positive result. Most bacteria and fungi can be grown in a variety of artificial media, but strict intracellular microorganisms (eg, Chlamydia, Rickettsia, and viruses) can be isolated only in cultures of living eukaryotic cells. The culture of some parasites is possible but used only in highly specialized laboratories. Isolation and Identification of Bacteria and Fungi Almost all medically important bacteria can be cultivated outside the host in artificial culture media. A single bacterium placed in the proper culture conditions multiplies to quantities sufficient to be seen by the naked eye. Bacteriologic media are soup-like recipes prepared from digests of animal or vegetable protein supplemented with nutrients such as glucose, yeast extract, serum, or blood to meet the metabolic requirements of the organism. Their chemical composition is complex, and their success depends on matching the nutritional requirements of most heterotrophic living things. The same approaches and some of the same culture media used for bacteria are also used for fungi. Growth in media prepared in the fluid state (broths) is apparent when bacterial numbers are sufficient to produce turbidity or macroscopic clumps. Turbidity results from reflection of transmitted light by the bacteria; depending on the size of the organism, more than 106 bacteria per milliliter of broth are required. The addition of a gelling agent to a broth medium allows its preparation in solid form as plates in Petri dishes. The universal gelling agent for diagnostic bacteriology is agar, a polysaccharide extracted from seaweed. Agar has the convenient property of becoming liquid at about 95°C but not returning to the solid gel state until cooled to less than 50°C. This allows the addition of a heat-labile substance such as blood to the medium before it sets. At temperatures used in the diagnostic laboratory (37°C or lower), broth–agar exists as a smooth, solid, nutrient gel. This medium, usually termed "agar," may be qualified with a description of any supplement (eg, blood agar). Page 1|8  Lec. 3 Microbial Diagnosis | Dr. Ahmad Hasan A useful feature of agar plates is that the bacteria can be separated by spreading a small sample of the specimen over the surface. Bacterial cells that are well separated from others grow as isolated colonies, often reaching 2 to 3 mm in diameter after overnight incubation. This allows isolation of bacteria in pure culture because the colony is assumed to arise from a single organism (Figure 4–7). Colonies vary greatly in size, shape, texture, color, and other features called colonial morphology. Colonies from different species or genera often differ substantially, whereas those derived from the same strain are usually consistent. Culture Media Over the last 100 years, countless media have been developed by microbiologists to aid in the isolation and identification of medically important bacteria and fungi. Only a few have found their way into routine use in clinical laboratories. These may be classified as nutrient, selective, or indicator media. Nutrient Media The nutrient component of a medium is designed to satisfy the growth requirements of the organism to permit isolation and propagation. For medical purposes, the ideal medium would allow rapid growth of all agents. No such medium exists; however, several suffice for good growth of most medically important bacteria and fungi. These media are prepared with enzymatic or acid digests of animal or plant products such as muscle, milk, or beans. The digest reduces the native protein to a mixture of polypeptides and amino acids that also includes trace metals, coenzymes, and various undefined growth factors. For example, one common broth contains a pancreatic digest of casein (milk curd) and a papaic digest of soybean meal. To this nutrient base, salts, vitamins, or body fluids such as serum may be added to provide pathogens with the conditions needed for optimum growth. Selective Media Selective media are used when specific pathogenic organisms are sought in sites with an extensive normal flora (eg, N gonorrhoeae in specimens from the uterine cervix or rectum). In these cases, other bacteria may overgrow the suspected etiologic species in simple nutrient media, either because the pathogen grows more slowly or because it is present in much smaller numbers. Selective media usually contain dyes, other chemical Page 2|8  Lec. 3 Microbial Diagnosis | Dr. Ahmad Hasan additives, or antimicrobics at concentrations designed to inhibit contaminating flora but not the suspected pathogen. Indicator Media Indicator media contain substances designed to demonstrate biochemical or other features characteristic of specific pathogens or organism groups. The addition to the medium of one or more carbohydrates and a pH indicator is frequently used. A color change in a colony indicates the presence of acid products and thus of fermentation or oxidation of the carbohydrate by the organism. The addition of red blood cells (RBCs) to plates allows the hemolysis produced by some organisms to be used as a differential feature. In practice, nutrient, selective, and indicator properties are often combined to various degrees in the same medium. It is possible to include an indicator system in a highly nutrient medium and also make it selective by adding appropriate antimicrobics. Atmospheric Conditions Aerobic Once inoculated, cultures of most aerobic bacteria are placed in an incubator with temperature maintained at 35° to 37°C. Slightly higher or lower temperatures are used occasionally to selectively favor a certain organism or organism group. Most bacteria that are not obligate anaerobes grow in air; however, CO2 is required by some and enhances the growth of others. Incubators that maintain a 2% to 5% concentration of CO2 in air are frequently used for primary isolation, because this level is not harmful to any bacteria and improves isolation of some. A simpler method is the candle jar, in which a lighted candle is allowed to burn to extinction in a sealed jar containing plates. This method adds 1% to 2% CO2 to the atmosphere. Anaerobic Strictly anaerobic bacteria do not grow under the conditions just described, and many die when exposed to atmospheric oxygen or high oxidation–reduction potentials. Most medically important anaerobes grow in the depths of liquid or semisolid media containing any of a variety of reducing agents, such as cysteine, thioglycollate, ascorbic acid, or even iron filings. An anaerobic environment for incubation of plates can be achieved by replacing air with a gas mixture containing hydrogen, CO2, and nitrogen and allowing the hydrogen to react with residual oxygen on a catalyst to form Page 3|8  Lec. 3 Microbial Diagnosis | Dr. Ahmad Hasan water. A convenient commercial system accomplishes this chemically in a packet to which water is added before the jar is sealed. Specimens suspected to contain significant anaerobes should be processed under conditions designed to minimize exposure to atmospheric oxygen at all stages. Appendix (1): Some Media Used for Isolation of Bacterial Pathogens MEDIUM USES General-Purpose Media Nutrient broths (eg, Soybean– Most bacteria, particularly when used for blood Casein Digest Broth) culture Thioglycolate broth Anaerobes, facultative bacteria Blood agar Most bacteria (demonstrates hemolysis) and fungi Chocolate agar Most bacteria, including fastidious species (eg, Haemophilus) and fungi Selective Media MacConkey agar Nonfastidious Gram-negative rods Hektoen-enteric agar Salmonella and Shigella Selenite F broth Salmonella enrichment Sabouraud's agar Isolation of fungi, particularly dermatophytes Special-Purpose Media Löwenstein–Jensen medium, Mycobacterium tuberculosis and other Middlebrook agar mycobacteria (selective) Martin–Lewis medium Neisseria gonorrhoeae and N meningitidis (selective) Fletcher medium (semisolid) Leptospira (nonselective) Tinsdale agar Corynebacterium diphtheriae (selective) Page 4|8  Lec. 3 Microbial Diagnosis | Dr. Ahmad Hasan Charcoal agar Bordetella pertussis (selective) Buffered charcoal–yeast extract Legionella species (nonselective) agar Campylobacter blood agar Campylobacter jejuni (selective) Thiosulfate-citrate-bile-sucrose Vibrio cholerae and V parahaemolyticus agar (TCBS) (selective) Appendix (2): Characteristics of Commonly Used Bacteriologic Media 1. Nutrient broths. Some form of nutrient broth is used for culture of all direct tissue or fluid samples from sites that are normally sterile to obtain the maximum culture sensitivity. Selective or indicator agents are omitted to prevent inhibition of more fastidious organisms. 2. Blood agar. The addition of defibrinated blood to a nutrient agar base enhances the growth of some bacteria, such as streptococci. This often yields distinctive colonies and provides an indicator system for hemolysis. Two major types of hemolysis are seen: β-hemolysis, a complete clearing of red cells from a zone surrounding the colony; and α-hemolysis, which is incomplete (ie, intact red cells are still present in the hemolytic zone), but shows a green color caused by hemoglobin breakdown products. The net effect is a hazy green zone extending 1 to 2 mm beyond the colony. A third type, γ-hemolysis, produces a hazy, incomplete hemolytic zone similar to that caused by α-hemolysis, but without the green coloration. 3. Chocolate agar. If blood is added to molten nutrient agar at about 80°C and maintained at this temperature, the red cells are gently lysed, hemoglobin products are released, and the medium turns a chocolate brown color. The nutrients released permit the growth of some fastidious organisms such as Haemophilus influenzae, which fail to grow on blood or nutrient agars. This quality is particularly pronounced when the medium is further enriched with vitamin supplements. Given the same incubation conditions, any organism that grows on blood agar also grows on chocolate agar. Page 5|8  Lec. 3 Microbial Diagnosis | Dr. Ahmad Hasan 4. Martin–Lewis medium. A variant of chocolate agar, Martin–Lewis medium is a solid medium selective for the pathogenic Neisseria (N gonorrhoeae and N meningitidis). Growth of most other bacteria and fungi in the genital or respiratory flora is inhibited by the addition of antimicrobics. One formulation includes vancomycin, colistin, trimethoprim, and anisomycin. 5. MacConkey agar. This agar is both a selective and an indicator medium for Gram-negative rods, particularly members of the family Enterobacteriaceae and the genus Pseudomonas. In addition to a peptone base, the medium contains bile salts, crystal violet, lactose, and neutral red as a pH indicator. The bile salts and crystal violet inhibit Gram-positive bacteria and the more fastidious Gram- negative organisms, such as Neisseria and Pasteurella. Gram-negative rods that grow and ferment lactose produce a red (acid) colony, often with a distinctive colonial morphology. 6. Hektoen enteric agar. The Hektoen medium is one of many highly selective media developed for the isolation of Salmonella and Shigella species from stool specimens. It has both selective and indicator properties. The medium contains a mixture of bile, thiosulfate, and citrate salts that inhibits not only Gram- positive bacteria, but members of Enterobacteriaceae other than Salmonella and Shigella that appear among the normal flora of the colon. The inhibition is not absolute; recovery of Escherichia coli is reduced 1000- to 10,000-fold relative to that on nonselective media, but there is little effect on growth of Salmonella and Shigella. Carbohydrates and a pH indicator are also included to help to differentiate colonies of Salmonella and Shigella from those of other enteric Gram-negative rods. 7. Anaerobic media. In addition to meeting atmospheric requirements, isolation of some strictly anaerobic bacteria on blood agar is enhanced by reducing agents such as L-cysteine and by vitamin enrichment. Sodium thioglycolate, another reducing agent, is often used in broth media. Plate media are made selective for anaerobes by the addition of aminoglycoside antibiotics, which are active against many aerobic and facultative organisms but not against anaerobic bacteria. The use of selective media is particularly important with anaerobes because they grow slowly and are commonly mixed with facultative bacteria in infections. Page 6|8  Lec. 3 Microbial Diagnosis | Dr. Ahmad Hasan 8. Highly selective media. Media specific to the isolation of almost every important pathogen have been developed. Many allow only a single species to grow from specimens with a rich normal flora (eg, stool). The most common of these media are are listed in Appendix 4–1; they are discussed in greater detail in following chapters. Appendix (3): Common Biochemical Tests for Microbial Identification 1. Carbohydrate breakdown. The ability to produce acidic metabolic products, fermentatively or oxidatively, from a range of carbohydrates (eg, glucose, sucrose, and lactose) has been applied to the identification of most groups of bacteria. Such tests are crude and imperfect in defining mechanisms, but have proved useful for taxonomic purposes. More recently, gas chromatographic identification of specific short-chain fatty acids produced by fermentation of glucose has proved useful in classifying many anaerobic bacteria. 2. Catalase production. The enzyme catalase catalyzes the conversion of hydrogen peroxide to water and oxygen. When a colony is placed in hydrogen peroxide, liberation of oxygen as gas bubbles can be seen. The test is particularly useful in differentiation of staphylococci (positive) from streptococci (negative), but also has taxonomic application to Gram-negative bacteria. 3. Citrate utilization. An agar medium that contains sodium citrate as the sole carbon source may be used to determine ability to use citrate. Bacteria that grow on this medium are termed citrate-positive. 4. Coagulase. The enzyme coagulase acts with a plasma factor to convert fibrinogen to a fibrin clot. It is used to differentiate Staphylococcus aureus from other, less pathogenic staphylococci. 5. Decarboxylases and deaminases. The decarboxylation or deamination of the amino acids lysine, ornithine, and arginine is detected by the effect of the amino products on the pH of the reaction mixture or by the formation of colored products. These tests are used primarily with Gram-negative rods. 6. Hydrogen sulfide. The ability of some bacteria to produce H2S from amino acids or other sulfur-containing compounds is helpful in taxonomic Page 7|8  Lec. 3 Microbial Diagnosis | Dr. Ahmad Hasan classification. The black color of the sulfide salts formed with heavy metals such as iron is the usual means of detection. 7. Indole. The indole reaction tests the ability of the organism to produce indole, a benzopyrrole, from tryptophan. Indole is detected by the formation of a red dye after addition of a benzaldehyde reagent. A spot test can be done in seconds using isolated colonies. 8. Nitrate reduction. Bacteria may reduce nitrates by several mechanisms. This ability is demonstrated by detection of the nitrites and/or nitrogen gas formed in the process. 9. O-Nitrophenyl- -D-galactoside (ONPG) breakdown. The ONPG test is related to lactose fermentation. Organisms that possess the -galactoside necessary for lactose fermentation but lack a permease necessary for lactose to enter the cell are ONPG-positive and lactose-negative. 10. Oxidase production. The oxidase tests detect the c component of the cytochrome–oxidase complex. The reagents used change from clear to colored when converted from the reduced to the oxidized state. The oxidase reaction is commonly demonstrated in a spot test, which can be done quickly from isolated colonies. 11. Proteinase production. Proteolytic activity is detected by growing the organism in the presence of substrates such as gelatin or coagulated egg. 12. Urease production. Urease hydrolyzes urea to yield two molecules of ammonia and one of CO2. This reaction can be detected by the increase in medium pH caused by ammonia production. Urease-positive species vary in the amount of enzyme produced; bacteria can thus be designated as positive, weakly positive, or negative. 13. Voges–Proskauer test. The Voges–Proskauer test detects acetylmethylcarbinol (acetoin), an intermediate product in the butene glycol pathway of glucose fermentation. Page 8|8