BACTERIOLOGY
METHODS OF IDENTIFICATION
The main concern in clinical bacteriology is the rapid and correct identification of pathogenic bacteria. Identification of the genus of a microorganism should be sufficient in many instances so that treatment is not delayed. Before studying any culture, it is important to know something about the growth requirements of the organism. But first, one must know the origin of the submitted specimen so that it can be inoculated in proper media. If the organism that is to be identified does not grow in ordinary media, it requires the complete absence of oxygen or of organic matter (or has other special requirements). Organisms that grow on ordinary media, methods must be varied according to whether the organism grow better in liquid or in solid medium and to low or high temperature. An organism may grow well on first isolation but in some instances cultures maybe adapted to the media used for routine testing of cultural properties through a series of transfers. There are different sets of conditions that will suit practically most bacteria, liquid and solid media at 37 C and at 21 C - 25 C. There are other organisms which require special growth requirement such as thermophilic bacteria like Campylobacter species.
Identification can be made by five general methods in clinical bacteriology:
1. Direct smear
- Gram stain
- Acid fast stain
- Fluorescent stain
- Special stain
2. Cultural methods
Solid media method:
- Streak plate - to isolate the organism in pure culture - most commonly used are Blood Agar Plate (BAP), MacConkey (MAC), Eosin Methylene Blue (EMB), Chocolate (CHOC), Hektoen (H), and Salmonella-Shigella (SS), Sabouraud agar (SAB) with or without antibiotics, Lowenstein-Jensen (LJ), and Middlebrook 7H10.
- Pour plate - to quantitate bacterial count in urine and blood
- Streak pour plate - to study hemolysis
Liquid media:
The inoculum is transferred by means of a sterilize loop or by directly inoculating the swab into the liquid. Wound specimens are inoculated into Thioglycollate broth (THIO); fecal specimen or rectal swab are inoculated in Selenite F broth (SF).
Incubation:
After the specimen has been properly inoculated, incubation should be done. Cultures for aerobic organisms growing best at body temperature are placed in an incubator which maintains a uniform temperature of 35 C - 37 C. Cultures for anaerobic organsims are placed in an anaerobe jar and incubated at 35 C - 37 C for 24 to 48 hours before opening the jar. Cultures to be grown in an atmosphere of 10% CO2 are placed in CO2 incubator at 35 C - 37 C or in candle jar. Cultures for fungi are grown at room temperature and incubator (35 C - 37 C) temperature. Most cultures are examined after 24 hours or 18 hours is enough.
3. Identification of bacteria
Solid Media:
- Colony characteristic - dry, mucoid, moist, rough, smooth, stippled, translucent, opaque, flat, elevated, shape of edges, swarming or spreading, satellite phenomenon and color.
- Odor produced - grapelike odor (Pseudomonas), mousey (Hemophilus), gun powdery (Proteus), fecal (Escherichia).
- Zone of hemolysis - Streptococci (alpha, beta, gamma); Pneumococci and Staphylococci may produce hemolysis.
Subculture:
In identifying Staphylococci, coagulase and mannitol tests are done.
Green hemolysis - Alpha Streptococci is differentiated from Pneumococci by Optochin disc. Optochin inhibits Pneumococci.
Clear hemolysis - Beta Streptococci Group A are differentiated from Group B, C, and D by its susceptibility to Bacitracin disc. Group B Streptococci by CAMP test - method developed by Australian researchers.
No hemolysis - Group D Streptococci (Enterococci) are positive in Streptococcus faecalis (SF) medium with acid reaction.
Neisseria spp. - a dark purple color in 5-10 seconds is positive for oxidase test (tetramethylparaphenyldiamine dihydrochloride). Other positive organisms are Pseudomonas, Mima, Herellea
Urine colony count:
Over 100,000 org/ml - indicative of urinary tract infection (UTI)
10,000 - 100,000 org/ml
1,000 - 10,000 org/ml
100 - 1,000 org/ml
No Growth (NG)
Liquid cultures:
Examine for turbidity, sediment, arrangement of colonies (floating, snowflake, chains), pellicle formation on surface, color development, gas and odor production, homogenous.
Biochemical reactions:
- Triple Sugar Iron agar (TSI)
- IMVIC - Indole, Methyl-Red, Voges Proskauer, Simmon-Citrate
- Motility tests - observe growth in or along the stab line.
- Gelatin liquefaction - make a gelatin stab, incubate and observe for rapid, slow or progressive liquefaction. The Salmonella Arizona group liquefy gelatin.
- Other biochemical tests are amino acids and sugars: Lysine decarboxylase, phenylalanine deaminase, lactose, sucrose, mannitol, dulcitol, salicin, adonitol and inositol.
4. Serologic identification
Based on the fact that flagellate bacteria possess not only a thermostable somatic O antigen but also thermolabile H antigen. Non-flagellate bacteria contain only the O antigen. A third antigen which is the Vi antigen in peripheral body is thermolabile in contrast to O antigen. It may interfere with agglutination of freshly isolated bacteria by anti-O sera.
The majority of Salmonella in the United States contain O antigen and can be identified by polyvalent O antisera. If positive agglutination occurs, proceed further to identify the group to which the organism belongs by using individual Salmonella O antisera Group A, B, C, and the other sera. If the culture reacts with Salmonella poly O antiserum, but does not react with the specific group, it should be checked with Vi antiserum. No agglutination Vi means the culture is not of genus Salmonella. If it reacts with Vi, it should be boiled for 30 minutes and cooled. The culture should be retested with the individual O antisera and Vi antiserum. If it does not react with Salmonella Vi antiserum but reacts with Salmonella O antiserum (Group D), it is most likely Salmonella typhi and this is confirmed using H antiserum Group d and unheated culture.
Typing of Shigella is more simply done than in the serological identification of Salmonella. Screening is carried out with polyvalent sera (A, B, C, D group). The technique is similar to Salmonella.
If Escherichia coli is found in infants, it is typed for known enteropathogenic serotypes of E.coli.
5. Identification by animal inoculation
Animals are used not only as an aid in the isolation of some bacteria in pure culture but also for the study of the pathology of infectious disease. The maintenance by animal passage of infectious agents, viruses, which can't be cultivated in media. The animals generally used are guinea pigs, rabbits, white mice and rats and in special cases: rhesus monkeys. The common routes of inoculation are intradermal, subcutaneous, intramuscular, intraperitoneal, and intravenous. Other routes are intraocular,, intracerebral and intrathecal. Inoculation is carried with a syringe and sterile hypodermic needle. The site of inoculation is prepared by removal of hair by shaving and cleansed with alcohol and tincture of iodine. The post mortem examination is determined by the particular disease under consideration, if this is not known, by the symptoms exhibited by the animal before death. Precaustion must be observed in autopsy of the sacrificed animal. If cultures are to be taken, fresh sterile instruments are used. Care must be taken to avoid disseminating infectious material, in highly contagious disease, such as tularemia. Instruments once used should be discarded or allowed to stand in a solution of lysol or cresol. Aseptic precautions must be exercised for infections of laboratory workers are not infrequent.
Published 9/16/11 altgroup multiply
Web Page: METHODS OF IDENTIFICATION
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