Objectives: When you have successfully completed all of the laboratory exercises, you will be able to:

1. Understand in a lasting, familiar fashion, the common human pathogenic bacteria and fungi that you will handle here (These exercises supplement and reinforce the lecture material.);
2. Perform and interpret Gram stains expertly and correctly;
3. Inoculate various clinical specimens onto the appropriate media for initial isolation of possible pathogens present;
4. Choose the best methods for procuring, transporting, and handling clinical specimens destined for the microbiology laboratory; and
5. Understand the capabilities and limitations of the diagnostic clinical microbiology laboratory.

Throughout these laboratory exercises, you will learn and practice both classical and contemporary procedures used by clinical microbiologists for isolating and identifying the common bacterial pathogens. These skills will be used by many of you throughout your entire professional careers. Another lasting gain from your laboratory experiences in this course will be your ability to evaluate the reports from clinical bacteriology labs.

Initially, pure cultures of various bacteria will be provided to acquaint you with the organisms most commonly isolated from patients in this country. You will perform various tests on these pure cultures to demonstrate important properties used in their identification. Handling these organisms in the lab gives them a reality that no amount of "book learning" can do. Specimens containing one or more "unknown" pathogens will be issued to you. These unknowns will provide an opportunity for you to test your newly acquired skills in isolating and identifying common human pathogens under relaxed (ungraded) conditions (you will be graded on your efforts made to identify the unknowns, not on the correctness of your conclusions). In general, the methods employed in these exercises are the same as (or similar to) those used in the University Hospital's Diagnostic Microbiology Laboratory and in diagnostic labs elsewhere in the world.

In general, all organisms found in specimens sent to a diagnostic laboratory are reported to the physician. The exceptions include normal flora (indigenous flora) found in stools or in throat cultures, depending on the particular clinical laboratory. It is then up to the physician to interpret the findings and take the appropriate action. When the specimen comes from a location in the body which normally is sterile (e.g., spinal canal, peritoneal cavity, pleural or joint spaces), it is usually a simple matter to interpret the laboratory results. When the specimen comes from an area which has its own normal flora (e.g., eye, nose, ear, throat, skin, stool, etc.), the physician must know the range and variability of the bacteria normally there before one can interpret the laboratory results -- e.g., a few staphylococci in these areas are normal; a massive number could indicate a staphylococcal infection.

Equally important is an awareness of the limitations of the bacteriology lab. The ability to recover and identify pathogens rapidly dwindles as the interval lengthens between the time a specimen is taken and the time it is plated onto media and properly incubated. Specimens should never be kept on a ward for several hours before being dispatched to the lab; nor should they be refrigerated in the hope of keeping the organism viable. Ideally, specimens should reach the lab or be plated within 20 minutes of the time they are collected. Anaerobes, the gonorrheal organism, and many others rapidly die once outside the body or if not properly incubated on suitable media. No aspect of this course has greater practical importance than your appreciation of the importance of the proper collection and processing of clinical specimens. The clinical lab can grow and identify from a specimen only what is viable. While we often think of bacteria as hearty organisms, this is not necessarily so for many pathogens.

Part of mastering a new subject is to become comfortable with the correct usage and spelling of its terminology. In the Microbiology part of IID, you will meet and need to learn the correct usage for naming microbes. Although an extensive hierarchy of terminology exists to designate the relatedness of groups of organisms, we will mostly be concerned only with the genus and species designations for each microbe, i.e., its binomial nomenclature. These are Latin names and, because Latin is a foreign language, they are italicized (e.g., Escherichia coli),or if underlined if handwritten (e.g., Escherichia coli). The genus (first of the two names) is comprised of a group of taxonomically very closely related organisms. This name is always capitalized. The species (second of the two names) is a subdivision of the genus comprised of organisms that differ by only very small characteristics. The species name is entirely lower-case (i.e., it is not capitalized). You will hear of strain differences within a species, and while these differences can have enormous consequences on the pathogenicity of the microbe, they represent only a few differences among the many, many properties that make up a living organism. It is common practice to write out the genus and species designation in full the first time it appears in text but subsequently to abbreviate the genus name by its first letter. Accordingly, Staphylococcus aureus becomes S. aureus, and Salmonella typhi becomes S. typhi. Even though there are many genus names that have common first letters, no confusion should arise, because most species names are unique. (In the few cases in which species names are identical, the genus first letters are different.)

For several bacteria, you will encounter a colloquial plural usage of the Latin genus name. In these cases, the designation is not italicized and not capitalized. Examples: staphylococci, streptococci, shigellae, salmonellae, yersiniae.

Here are singular and plural forms of some Latin or Latin-derived words that you will be encounter.

Singular Plural

1. Words ending in -um or -a

as in:

bacterium bacteria

serum sera

septum septa

flagellum flagella

2. words ending in -us or -i

as in:

bacillus bacilli

terminus termini

pilus pili

$ Exception…

genus genera

3. words ending in -a or -ae

as in:

petechia petechiae

macula maculae

Salmonella salmonellae

Shigella shigellae

It is VERY IMPORTANT BEFORE EACH LABORATORY PERIOD to CAREFULLY READ the exercise(s) to be covered that day and to review the lecture material pertaining to the exercise(s). Any student who comes to the laboratory "cold" may not only have difficulty completing the work in the allotted time, but may not understand as well the significance of what she or he is doing.

1. Mouth pipetting is not allowed. Mechanical pipetting devices (bulbs or pumps) are provided for this purpose.
2. Long hair should be tied back to prevent contact with cultures and Bunsen burners.
3. In the case of a personal accident, e.g., cuts, finger pricks, spray in eyes, etc. that may lead to infection, report to the laboratory instructor immediately.
4. Cover open cuts, broken skin, or dermatitis on hands with a band-aid or other suitable means such as latex or vinyl gloves.
5. All cultures, which are to be incubated, should bear the following information:

a. Name of student

b. Specimen identification

c. Date

There are three main areas for discard:

$ To clean up a spill: Gently place one or more paper towels over the spill. Then soak the contaminated area with disinfectant and allow to soak for at least 15 minutes before being cleaned up and discarded.

1. Clean oil off the oil immersion lens of your microscope at the end of each laboratory period. Use lens paper, only—do not use paper towels, bibulous paper, or other "scratchy" paper.

The following equipment and supplies are some of the items needed in the course:

Laboratory coat

Lens paper

Bibulous paper

Microscope slides

Glass marker (wax pencil)

Use the same lab station in MS 233 (2nd floor Histology Lab) you were assigned last year. Remember that you are responsible for the security of your microscope; keep it locked up when you're not using it. You are responsible for returning other assigned materials and removing personal items and debris from your lab station at the end of the course—failure to do so will result in a 1% penalty deduction from your final numerical score for the course.

As a convenience for you, we have provided two appendices at the end of this manual. Appendix 1, The Bacteria, summarizes the simplified identification schemes you will use to identify unknowns and compiles descriptions of the bacteria that you have met during the bacteriology laboratory exercises. Appendix 2, the Culture Media and Biochemical Tests, is a compilation of brief descriptions of the media and diagnostic tests that you will meet during the bacteriology laboratory exercises.

The laboratory room has been subdivided into 5 sectors according to the diagram on the next page. Note which sector you are in and who your primary instructors will be for the lab.

 

 

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