Clinical Laboratory Microbiology A Practical Approach Pdf Files

INTRODUCTION The purpose of the Clinical Microbiology course for undergraduate Medical Laboratory Science students is to gain familiarity with medically important bacteria and fungi by performing the full range of clinical laboratory tests in microbiology. Many clinical specimen samples, such as stool, contain numerous organisms, but Clinical Microbiology professors may be limited to single-organism specimens in teaching due to the complexity of creating reproducible mixed cultures. To assist professors in training students in the crucial skills of isolating pathogenic bacteria from normal flora to be properly prepared for work in the hospital laboratory, we present protocols to develop reproducible, mixed cultures for educational training. This article includes detailed protocols for creating mock stool, urine, wound, and throat cultures for academic laboratory use.

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These cultures are useful as advanced laboratory activities, after students are confident in skills of processing, plating, and identifying organisms in isolation () (, ). Ideally, students would have multiple laboratory sessions in a single week for this exercise, to independently follow cultures from sample processing to clinical report; however, instructions are included for preparing the samples and having students evaluate grown cultures for the presence of normal flora and pathogens in a single laboratory session. The protocols specify concentrations and amounts of organisms to combine, as well as how to present samples as though students were receiving unknown patient samples in a clinical setting. Protocols are provided for creating clinical samples with normal flora, contamination (indicating poor sample collection), and pathogens in the presence of normal flora and contamination. The cultures contain two to four organisms and take approximately three days to prepare for student use. Required materials The general protocol is described here, and specific protocols for wound, stool, urine, and throat samples are provided in the.

In addition to the relevant organisms, required materials include sterile plastic tubes with screwcap lids, sterile 5-mL pipettes, micropipettes and tips, a spectrophotometer, sterile cuvettes, a CO 2 incubator or appropriate container, sheep blood agar (SBA) plates, additional agar plates appropriate to the site of interest (see for suggestions), sterile inoculation loops, and prepared tryptic soy broth (TSB). For mixed cultures in a non-clinical laboratory, other agars, such as trypticase soy agar, may be used, but the colony morphologies may not be distinct between organisms. Topics to discuss in class prior to the laboratory experience Patient samples submitted to the clinical laboratory must be evaluated for quality of the specimen as well as the presence of pathogens. Poorly collected specimens, especially skin wounds and urine samples, will show “contamination” by normal skin flora (–), and a new sample submission to the laboratory may be required. Other patient specimens, such as stool samples, contain an abundance of normal flora, and the challenge is to identify the presence of a pathogen amongst the normal flora. Only the pathogen(s) should be isolated and identified to report to the clinician.

Finally, it is possible to have more than one pathogen in a single patient specimen, with or without normal flora. It is important in these circumstances to isolate and identify all pathogens for the clinician. While the multi-organism cultures reported here are not as complex as some clinical specimens, they provide realistic, introductory examples to aid students in understanding the difficulties encountered with patient specimens in the clinical laboratory. PROTOCOLS Instructor and student protocols specific to specimen sites can be found in the; a general workflow is shown in.

Once the instructor has determined the specimen type (wound, stool, urine, or throat) and the mixed culture type (contamination only, pathogen and contamination, mixed pathogens), the can be used to identify organisms for the culture. Each organism is grown separately in TSB and diluted to the specified spectrophotometer readings to estimate the amount of organism present. The organisms can then be combined in appropriate ratios to create mock clinical samples. Two options for presenting the samples to students are included, based on whether the students will interact with the samples over one or multiple laboratory sessions. If one session is used, instructors should culture the organisms using suggested media (see ) and incubate prior to class.