Collection of Urine Specimen

Immediately upon entering lab, check with teacher.  You will:

1.                  Be assigned to collect either a voided (“casual catch”) urine or a “clean catch, mid-stream” urine.

a.      For voided urine, simply urinate into cup and bring cup back to lab.

b.      For clean catch,

                                                              i.      Use antiseptic wipe to thoroughly clean area around urethral opening (the “clean” of clean catch)

                                                            ii.      Urinate into toilet—this allows any bacteria in lower part of urethra to flow out

                                                          iii.      After initial stream has continued for a while, urine specimen may be collected (this is the “mid-stream” of the collection) and brought to lab

2.                  Upon return to lab, label three plates with your name and the words “early” and “urine.”

a.      PEA

b.      TSA (blood agar)

c.      MacConkey’s

3.                  Using a special sterile, calibrated loop, dip into urine and streak a plate from top to bottom (see illustration), with streak lines very close together at the top, becoming further and further apart as you progress down the plate (you are NOT streaking for isolation here!):

4.                  Repeat with fresh loopful for each plate.

5.                  Do NOT dispose of urine at this time.  Replace cap and allow to sit on desk.

6.                  Place plates in “incubate” tubs.

7.                  Return to desks for quiz.

Completion of Skin, Oral, and Respiratory Sub-Cultures

Perform required biochemical tests (e.g., coagulase, bacitracin, optochin testing) on subcultures.

Coagulase:

1.      Obtain test tube containing a measured 0.5 mL of rabbit plasma.

2.      Using a sterilized inoculating loop, place a colony (or more) into this broth.

3.      Incubate the test tube at 35-37 degrees.

4.      At four hours, examine for the presence of a clot (positive for S. aureus) and then re-examine again at 24 hours if negative at four hours (lab assistants will remove tubes after 24 hours).

Catalase:

1. Place a drop of hydrogen peroxide on a clean glass slide.
2. Using a sterile inoculating needle, mix a selected colony with the peroxide.
3. The presence of bubbles of oxygen is indicative of a positive (Staphylococcus species) reaction.

Oxidase:

1.      Place an oxidase strip on a paper towel.

2.      Using a sterile inoculating loop, scrape part of your bacterial colony onto the oxidase strip.

3.      A blue/purple color change is positive for Neisseria species or Pseudomonas.

Bacitracin:

Disks impregnated with bacitracin are placed on blood agar plates containing Streptococcus.  After incubation, the following patterns can be observed:

The main purpose of this test is to presumptively identify Group A Streptococcus.

Optochin:

This test is used to presumptively differentiate Streptococcus pneumoniae from other alpha hemolytic Streptococci.  Because S. pneumoniae is susceptible to extremely small concentrations of the antibiotic, optochin (unlike the other Streptococcus, which are susceptible only to larger amounts), a disk containing minute amounts of optochin is placed on a blood agar plate with presumptive S. pneumoniae.  After incubation, if there is a zone of inhibition that indicates susceptibility to optochin, S. pneumoniae are presumptively identified.

Normal Flora of the Gastrointestinal Tract

The digestive system is divided into the gastrointestinal (GI) tract, also known as the alimentary canal, and the accessory digestive structures, which, except for the teeth and tongue, are external to the alimentary canal. 

GI Tract:

·        Mouth

·        Pharynx (throat)

·        Esophagus

·        Stomach

·        Small intestine

·        Large intestine

Accessory Digestive Structures:

·        Teeth

·        Tongue

·        Salivary glands (produce secretions carried by ducts)

·        Liver (produces secretions carried by ducts)

·        Gallbladder (stores secretions carried by ducts)

·        Pancreas (produces secretions carried by ducts)

Normal flora are found in the

·        Mouth (many)

·        Stomach (relatively few)

·        Small intestine (relatively few)

·        Large intestine (many)                                                                                         

When Transient Flora Go Sightseeing

Expected Normal Flora

Small intestine:

Candida albicans

Streptococci, Lactobacilli, diptheroids

Large Intestine:

Anaerobic Gram negative bacteria

·        Bacteroides

·        Prevotella

·        Fusobacterium

Anaerobic Gram positive bacteria

·        Clostridium perfringens and other species

·        Peptostreptococcus

·        Peptococcus

Facultative anaerobic Gram negative rods (the coliforms)

·        Escherichia coli

·        Klebsiella species

·        Enterobacter species

·        Proteus species

Others

·        Enterococcus species (facultative anaerobe, Gram positive cocci)

·        Staphylococcus aureus in carriers (facultative anaerobe, Gram positive cocci)

·        Pseudomonas species (aerobe, Gram negative rods)

·        Candida species (fungi)

Bacterial Diseases of the GI Tract

Either due to:

A.                 Actual colonization of organisms within the intestinal tract (“infection”)

                                                              i.      Once established as infections, the bacteria may secrete enterotoxins that interfere with intestinal tract functions (e.g., cholera, bacillary dysentery, salmonelloses)

                                                            ii.      Infections are characterized by a delay in the appearance of GI symptoms while the pathogen increases in numbers and affects the invaded tissues (12 hours to 2 weeks)

                                                          iii.      Fever is generally associated with the infection

B.                 Effects of pre-formed bacterial toxin ingested with food (“intoxication”)

                                                              i.      These are classified as food poisonings (e.g., botulism, staphylococcal food poisoning)

                                                            ii.      Characterized by very sudden onset, usually within a few hours, of GI symptoms

                                                          iii.      Fever is not generally associated with intoxication

Review of Rectal Isolates:  Compare your colonies to the colonies of the known bacteria, E. coli, Enterococcus faecalis, K. pneumoniae, P. mirabilis, Enterobacter cloaceae, and Salmonella enteritidis.

1.                  Look at the blood agar plate first.

a.      Recall, this is both basic and differential medium.

b.      Note the number and variety of colonies.

c.      Note the presence of any hemolysis.

2.                  Look at the MacConkey’s plate next.

a.      Recall, this is both selective (for Gram negative organisms) and differential (lactose fermenters turn pink).

b.      Both Enterobacteriaceae and non-Enterobacteriaceae will grow on MacConkey’s—determine which you have by performing an oxidase test (Enterobacteriaceae are fermenters and, thus, are oxidase negative).

c.      You may want to review the Dichotomous Scheme for further clarification.

d.      Perform a Gram stain from the MacConkey’s.

3.                  Look at the Hektoen plate next.

a.      Recall, this is selective for enteric organisms (those organisms that live within the intestines) and differential for lactose fermenters (turn orange/yellow; non-fermenters remain the same color as the medium) and for those that produce H₂S (turn black)

b.      Thus, lactose fermenters (which are generally non-pathogenic) are orange/yellow

c.      Non-fermenters, which MAY be pathogenic, are green

d.      H₂S-producers, which may or may not be pathogenic, are black

4.                  Finally, look at the PEA plate.

a.      Recall that PEA is selective for Gram positive organisms and differential, according to hemolysis.

b.      Except for Enterococcus, you should not have much growth here (if you do, then you did not collect this specimen properly and may have skin contaminants present!).

c.      Perform a catalase test on anything growing on the PEA.  Note that Enterococcus is often beta hemolytic on PEA.

5.                  Using the Dichotomous scheme, select an isolated colony from the MacConkey’s plate for further identification.

a.      Inoculate carbohydrate broths with Durham tubes

b.      Inoculate Simmon’s citrate agar slants

c.      These will be read next week.

6.                  In addition, two isolates will be selected from the class to be streaked on blood agar and subsequently set up using the API system.

Before You Leave!

1.      Label three more plates with your name and the words “late” and “urine.”

a.      PEA

b.      TSA (blood agar)

c.      MacConkey’s

2.      Inoculate these, as before.

3.      Take urine to bathroom, pour into toilet, and flush.

4.      BRING CONTAINERS BACK FOR DISPOSAL IN BIOHAZARD BAG.

Gram Negative and Positive Dichotomous Schemes

Gram Stain

Instructions for Gram staining are re-printed below

Materials:

Inoculating loop

Glass slide

Slide holder

Glass marking pen

Bunsen burner

Flint lighter

Bibulous paper

1.         Label your slide, if you plan on keeping it.

2.         Place a small drop of .85% saline on the slide.

3.         Sterilize your inoculating loop and select a colony.  Pick it up and mix it with the saline.

4.         Allow the saline/colony solution to dry fully.

5.         Heat fix it.

6.         Gram stain it:

            a.         Crystal violet, one minute.  Rinse with water.

            b.         Iodine, one minute.  Rinse with water.

c.         Alcohol, until runoff is clear OR 20 seconds, whichever is shorter.  Rinse with water.

d.         Saffranin, one minute.  Rinse with water.

7.         Blot dry, using bibulous paper.

8.         Coarse focus on 10x; fine focus, using oil, on 100 x.

Take Out Food for the Brain:

Hepatitis, a term that describes the inflammation of the liver, can be due to a variety of causes.  It can be caused by drug or chemical toxicity, an autoimmune response, or it can be infectious in nature.  Bacteria, parasites, and viruses have all been implicated in hepatitis, but by far, viral hepatitis is of the greatest significance, placing enormous demands upon economic and medical resources.  The control of viral hepatitis ultimately depends upon the application of two important pieces of information:

• The awareness of known and potential risks for acquiring the different types of viral hepatitis infections
• The awareness of preventive and therapeutic measures for the different types of viral hepatitis infections

In addition, for the health care worker, knowledge of risk behavior histories of patients is helpful, and an understanding of the appropriate diagnostic tests that are available for the different types of viral hepatitis is essential.

The three major causes of viral hepatitis are listed below, along with some important information (three other viral causes, HDV, HEV, and HGV, are not discussed here; of these, HDV also requires the presence of active HBV infection before it can cause further infection).

Hepatitis A

Etiologic agent:  Hepatitis A virus (HAV)

Transmission:  person-to-person, by fecal contamination and oral ingestion.  Transmission may occur through intra-household or sexual contact if personal hygiene or sanitation is poor.  Transmission may also occur through intravenous drug use.  Contaminated food and water can create epidemic situations.

Vaccine available.

Hepatitis B

Etiologic agent:  Hepatitis B virus (HBV)

Transmission:  percutaneous or permucosal; infective blood/body fluids can be introduced at birth, through sexual contact (predominant mode of transmission), or by contaminated needles

Vaccine available.

Hepatitis C

Etiologic agent:  Hepatitis C virus (HCV)

Transmission:  parenterally, through blood and blood products, transplanted organs, and intravenous drug abuse; may be sexually transmitted, although not as efficiently as parenteral route; perinatal transmission is unclear, but probably occurs.

No vaccine available at this time.

Serodiagnosis of Hepatitis

Take Home Thought

Chronic liver disease is the tenth leading cause of death in the U.S., and about half of these deaths are due to HCV.  According to the CDC, HCV infection will eventually be responsible for more deaths than AIDS.

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