Clinical Bacteriology Lab - 10 Unknown Bacteria Identification and Miscellaneous Procedures

CLINICAL BACTERIOLOGY LABORATORY PROCEDURE INDEPENDENT IDENTIFICATION OF UNKNOWN BACTERIA AND MISCELLANEOUS PROCEDURES 1. OUTLINE • • • • • 2. Introduction Identification of Unknown Bacteria Blood Culture Urine Culture Water Analysis • INTRODUCTION • • • • • One important concern of Medical Microbiology is to isolate and identify the causative organism of a disease and in doing this, mastery of technique is necessary for accurate microbial isolation and identification. In the process of microbial identification, all knowledge and skills in microbiology must be completely understood because the students are allowed to plan their own laboratory protocol with emphasis on aseptic technique. Observation of aseptic technique must be observed at all times to avoid microbial contamination. This technique is necessary in the inoculation of microorganisms, preparation of culture media and dispensing of medium to appropriate container. One must bear in mind that contamination from air and other source cannot be easily monitored in the laboratory; therefore, care should be taken in every step done in the process of isolation and identification. Proper use of staining methods and culture media will make individual protocol easy to perform. For this crucial selection of procedure, avoid complication and discrepancy of result. To review some important methods which include: (1) Gram staining which group bacteria according to their cell wall composition (gram positive and gram negative), morphology and arrangement, this procedure will allow microbiologists to decide which further steps are needed, (2) Use of appropriate culture media (selective, differential and special) provide an easily isolation of intended organism and lastly, (3) the use of important biochemical test facilitate a reliable microbial identification. One must bear in mind that other method and procedure must be considered such a serological test, animal inoculation, phage typing, amino acid sequencing, protein analysis and DNA base composition are other advanced methods for a successful microbial identification. 3. 4. Gram staining of the unknown bacteria is performed. Besides gram staining, its morphology and arrangement is also recorded. Motility test of the bacteria is determined by hanging drop preparation (Figure 7.25). Using sterile inoculating technique, the bacteria is inoculated into two nutrient agar slants, a nutrient broth tube and to a nutrient agar plate by means of streak inoculation. Following incubation, one slant culture is used to determine the cultural characteristics of the unknown bacteria. The second is used as a stock sub-culture, should it be necessary to repeat any of the tests. Growth characteristics are also observed in the broth tube and colony characteristics on the plate. Exercising care in sterile technique, so as not to contaminate cultures and thereby obtain spurious results, the bacteria is inoculated into respective media to carry out the different biochemical tests. The inoculated media are incubated at the required temperatures for the required lengths of time. IDENTIFICATION OF UNKNOWN BACTERIA MATERIALS & PROCEDURES OBSERVATIONS & INTERPRETATION OF RESULTS MATERIALS REQUIRED • Slides, cavity slides, petri dishes, conical flasks, cotton plugs, inoculating loop, autoclave, bunsen burner, laminar flow chamber, dispose jar, incubator, nutrient broth, nutrient agar, gram staining reagents, media and reagents for biochemical tests, compound microscope, isolated colonies or pure cultures of bacteria. • Observations: o In gram staining, besides gram reaction, the morphology and arrangement of the bacteria are also recorded. o The cultural characteristics of the bacteria in nutrient broth, on agar slant and on nutrient agar plate are noted. o Results of the biochemical reactions are recorded. o • Interpretation of Results: o Using the above recorded data (For example, see Table 7.1) and with the help of the Bergey’s Manual of Determinative Bacteriology, the bacteria is identified as to its genus and species. It should be kept in mind that, results might vary depending on the strains of each species used and the length of time the bacteria has been maintained in stock culture. URINE CULTURE INTRODUCTION RATIONALE • BLOOD CULTURE INTRODUCTION • • • Blood for culture is probably the most important single specimen submitted to the microbiology laboratory for examination. It helps provide a clinical diagnosis as well as specific etiologic diagnosis. The presence of microorganisms in the patient’s blood reflects active and possibly spreading infection in the tissues. The prognosis of such bacteremia or septicemia may depend on its prompt recognition by bacteriologic means. Subsequent initiation of specific therapy based on laboratory findings may prove to be lifesaving. Likewise, a negative culture would prove helpful in ruling out microbial etiology. Primary pathogens: Salmonella, Brucella, other Gramrods, Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae NOT pathogens unless recovered from > 1 separate blood culture: Bacillus spp., coagulase(-) staph, viridans Streptococci, coryneform Gram (+) rods • • • • PRINCIPLE • • Materials o Blood specimen (sterile) o BAP, Chocolate agar and Thioglycolate broth o Candle jar Procedure 1. Extract 10 mL blood aseptically. 2. Transfer 5 mL blood to a thioglycolate broth and the other 5 mL to a bottle with BHI agar and BHI broth. o Note: The thioglycolate broth is boiled for 10 minutes before use to lessen the oxygen content. 3. Incubate 37 degrees Centigrade for 24 hours. 4. Streak the BAP and chocolate agar, incubate the BAP at 37 degrees Centigrade and the chocolate agar in a candle jar at 37 degrees Centigrade. 5. Perform biochemical testing for microbial identification Urine cultures are evaluated with consideration given to factors such as colony count, type and number of organisms growing, type of specimen and the clinical condition of the patient. MATERIALS & PROCEDURES MATERIALS & PROCEDURE • Urinary tract infections (UTI) are one of the most commonly encountered acute infectious diseases. Most UTIs occur as a result of bacteria ascending the urethra and entering the urinary bladder. Urine specimens for culture are collected when the following syndromes are suspected: cystitis, pyelonephritis, asymptomatic bacteriuria, and less commonly acute prostatitis, pyelonephric abscess, and urosepsis. Among the bacteria most commonly isolated from patients with acute uncomplicated cystitis are Escherichia coli, Klebsiella species, and other Enterobacteriaceae and Staphylococcus saprophyticus. Group B Streptococcus are markers of colonization in pregnant women. • • Materials o Compound Microscope o Sterile wide-mouthed container o MacConkey agar plate o Blood Agar plate o Calibrated inoculating loop o Alcohol lamp o Midstream catch urine specimen Procedure 1. Wash and collect a morning midstream catch urine a sterile container. o Wipe the outside of the container with disinfectant and close it tightly. 2. After collection, bring to the laboratory and should be processed within 1 hour after collection. 3. If it cannot be processed immediately, urine should be kept in the refrigerator at 4OC and must be processed not longer than 18 hours. 4. 5. 6. 7. 8. 9. Transfer approx. 1.0 mL of urine sample in a small sterile test tube. Hold the tubes to the light and examine urine for color, turbidity and pH. Note the odor of the urine. Using the calibrated loop, get a loopful of the urine sample (from the original container) and streak onto the BAP. Repeat step #5 and streak onto the MacConkey agar plate. Incubate all plates at 35OC to 37OC for 24 hours. Examine the plates for any growth. Count the colonies on BAP and report the number of organisms per milliliter. PROCEDURES • • WATER ANALYSIS INTRODUCTION • • • The importance of water contamination in the spread of infections is so well recognized and controlled in most countries by public health measures that it is difficult for most of us to realize how serious water contamination can be. Unfortunately, epidemics of water borne diseases such as cholera, dysentery, and typhoid fever still occur in parts of the world today and these diseases could easily become epidemics if not rigidly controlled. Water provides a habitat for many different microorganisms. Among bacteria most commonly found in natural waters are sulfur bacteria, iron bacteria, free living spiral forms, and spore-formers. Since water is in contact with the soil, many common soil inhabitants, such as Bacillus subtilis are usually present. However, the important facts to remember about the flora of water are the pathogenic microbes. These microorganisms should not be present in drinking water for human consumption. Hence, water that contain pathogenic organism is regarded as fecally contaminated. A test for fecal contamination is accomplished by looking for an organism which occurs only in feces and never as free-living in nature. There are several microbes which are used as indicator for fecal contamination which include, Escherichia coli, Streptococcus faecalis, Clostridium species and Candida albicans. The findings of these microbes are sufficient evidences that the water is not safe for drinking purposes and therefore suggest fecal contamination. METHODS AND MATERIALS • Materials o Water samples from different sources o Inoculating loop o Fermentation tubes o Plated EMB o Colony counter o Sterile pipettes (1ml and 10 ml) o Alcohol lamp o Sterile water o Nutrient agar • 1st Day- Colony Counting 1. Prepare serial dilution of the water sample, 1:10, 1:100, up to 1:1,000,000 using sterile distilled water. 2. Pipet 1 ml of each dilution in a sterile plate starting from 1:1,000,000 or to 1:10. 3. Pour about 15 ml of the agar into the plate with diluted water. Mix by swirling and allow to solidify. 4. Incubate at 37◦C for 24 hrs. 5. Count the number of colony and report it as colony forming units/ml(CFU/ml) 1st Day - Presumptive Test 1. Pipet 1 ml of water sample to test a tube with inverted fermentation tube containing 9ml Lactose broth. 2. Incubate at 37◦C for 24 hrs. 3. Observe tubes for gas production. A positive presumptive test is one with gas formation. 4. If tube is without gas, stop the analysis. If there is gas production, proceed to confirmatory test. 2nd Day - Confirmatory Test 1. Streak EMB plate with organism from the test tube with gas production. 2. Incubate at 37◦C. 3. After incubation observe for: ▪ Escherichia coli - small colonies with greenish metallic sheen. ▪ Enterobacter specie - large pinkish mucoid colonies with dark centers. ▪ Enterobacter aerogenes - large colonies with brown red center. 4. A positive confirmatory test constitutes a growth of typical colonies. 5. Proceed to completed test. • • • 3rd Day - Completed Test 1. Inoculate the distinct colonies to a fermentation tube with lactose broth. 2. Incubate at 37◦C. 3. Make a gram stain and observe for the presence of gram negative, non-spore forming bacilli. 4. After incubation, observe for a gas formation in lactose broth. A positive completed test is the production of gas in lactose broth and a gramnegative non-spore forming bacilli. 5. To differentiate the fecal from the non-fecal contaminations, inoculate the isolated bacteria to SIM, MR-VP, Simon citrate for the IMViC test. 4th Day - Reading of Biochemical Test 1. After 24 hour incubation at 40◦C, read the biochemical test by adding appropriate reagents to the different culture media.