James O'Connor, MS, MT (ASCP)*
James O'Connor, MS, MT (ASCP)
1. Group A Streptococcus pyogenes is the etiological agent of streptococcal pharyngitis, scarlet fever, pyoderma and many other disease processes. If untreated, a small percentage of the infected patients may develop serious sequelae infections, such as, rheumatic fever and post streptococcal glomerulonephritis. It is important that the infected patient be rapidly identified so that appropriate antibiotic prophylaxis can be initiated.
2. A properly and adequately obtained swab of the posterior pharynx is essential. There should be complete physical contact of the swab with inflamed areas, vesicles and pustular tonsils.
3. Most of the commercial kits on the market are based on the
principle of latex particle agglutination or enzyme linked
immunosorbent assay (ELISA). It is very important that all the
manufacturers directions be explicitly followed. The first step in
these assays is to extract the group A specific carbohydrate
antigen from the cell wall of the organisms in the throat swab.
Nitrous acid, Pronase B enzyme, hot formamide, hot HCL, and other
solutions can be used for the extraction process. Extraction times
range from 5 minutes to 1 hour depending on the extraction solution
utilized. In the latex particle agglutination procedures a drop of
throat swab extract is added to a circle on a glass slide containing
latex particles adsorbed with specific antibody to group A
streptococcus carbohydrate antigen. The slide is rotated for several
minutes and examined for agglutination (Positive test) 
(Figure
1). A negative control can also be included and consists of 1
drop of extract added to a circle containing latex particles adsorbed
with normal rabbit immunoglobulin. No agglutination (negative test)
should be obtained. A known positive control (group A Streptococcus pyogenes cells) should also be included and
treated in the same manner as the throat swab to ensure good quality
control. In the ELISA procedures a plastic template with a
funnel-shaped oriface contains a cellulose acetate membrane. Specific
antibody to group A streptococcal carbohydrate antigen is bound to
the membrane. The throat swab extract is added to the membrane. If
group A streptococcal antigen is present it is "captured" by the
immobilized antibody and sticks to the membrane. After washing with
buffer, goat anti-group A streptococcal antibody conjugated with
enzyme (e.g. horseradish peroxidase) is added to the membrane. This
conjugate binds to the membrane bound streptococcus antigen. After
washing with buffer, a substrate for the enzyme is added and the
formation of a color (purple, yellow etc. depending upon substrate)
indicates a positive test. Absence of color formation on the membrane
is a negative test.
4. These rapid strep identification kits have been reported to be very specific (healthy people give a negative test). Sensitivity reports have ranged from approximately 60% sensitivity to greater than 95%. Since false negatives can be obtained, all negative rapid strep ID tests should be followed by culture of the throat swab on a blood agar plate for the isolation of beta hemolytic streptococci. Positive tests do not require culture follow-up.
James O'Connor, MS, MT (ASCP)
Circle A shows a positive test (agglutination of the latex particles.) The other circles B-E show negative tests (no agglutination of the latex particles).
James O'Connor, MS, MT (ASCP)
1. Needless to say it is important that the specimens be obtained properly and smears prepared as soon as possible. Smears can be made with a swab or if liquid a pipet can be used. The smear should be about the size of a nickel and contain both thick and thin areas. Allow to air dry.
2. The Gram stain procedure can be found in all standard microbiology textbooks. Table 1 shows the procedure for the rapid Gram stain method. Gram positive and gram negative results are due to differences in the chemical structure of the bacterial cell wall. Gram positive organisms have a thick peptidoglycan layer and less lipid and thus retain the crystal violet primary stain and stain purple (don't decolorize). Gram negative organisms have a thinner peptidoglycan layer and a lot of lipid in cell wall. The crystal violet is removed by the decolorization procedure. They then stain red with the safranin counterstain. The purpose of the Gram's iodine (the second solution in the procedure) is to act as a mordant. It enables the crystal violet to fix more strongly to the cell wall of gram positive organisms. The decolorization procedure is the most important step in the Gram stain. The acetone-alcohol decolorizer should be applied for only 5-10 seconds (or until no more blue color comes out with the decolorizer wash). Some organisms easily over decolorize (e.g. streptococci). It takes practice to acquire good gram stained results.
3. In a properly stained smear the cytoplasm and nuclei of
segmented neutrophils should be pink to red--not purple. Quality
control of the Gram stain procedure can be carried out by
incorporating known organisms, such as, Staphylococcus aureus (gram positive cocci) and E. coli (gram negative rods) along
with the patients smears to insure proper performance of the
procedure. If these quality control organisms are not available,
smears of gingival scrapings can be gram stained. If both gram
positive and gram negative bacteria are present it indicates a good
gram stained smear 
(Slide
#1). If all organisms are purple the slide has been under
decolorized 
(Slide
#2). If all organisms are pink to red, over decolorization has
occurred 
(Slide
#3).
4. In gram stained smears of pus from boils and abscesses one
would especially be looking for the presence of staphylococci which
appear as gram positive cocci in grape-like clusters 
(Slide
#4) or streptococci (gram positive cocci in singles, pairs and
chains - the cocci can be somewhat elongated). Segmented neutrophils
should stain pink to red 
(Slide
#5): Red blood cells will stain blue in a properly prepared Gram
stain.
5. Middle ear infections are most commonly caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. In tympanocentesis fluid Streptococcus
pneumoniae appear as gram positive lancet-shaped diplococci ("ants
without legs") 
(Slide
#6). Haemophilus influenzae are seen as thin pleomorphic
gram negative rods to coccobacilli. Occasional filamentous forms may
occur 
(Slide
#7). Moraxella catarrhalis is a gram negative diplococcus
with adjacent sides flattened (look like kidney beans). Segmented
neutrophils should stain pink to red 
(Slide
#8).
6. Conjunctivitis or pink eye is most commonly caused by Haemophilus influenzae, Streptococcus pneumoniae and Staphylococcus aureus. In gram stained conjunctival smears these organisms would appear the same as previously described in tympanocentesis fluid and pus from abscesses.
James O'Connor, MS, MT (ASCP)
Gingival Scraping -- Both gram positive and gram negative bacteria Section Top | Title Page
James O'Connor, MS, MT (ASCP)
Gingival Scraping -- all bacteria are purple (underdecolorized) Section Top | Title Page
James O'Connor, MS, MT (ASCP)
Gingival Scraping -- all bacteria are red (overdecolorized) Section Top | Title Page
James O'Connor, MS, MT (ASCP)
Staphylococci in pus (gram positive cocci in grape-like clusters) Section Top | Title Page
James O'Connor, MS, MT (ASCP)
Streptococci in pus (gram positive cocci in singles, pairs and chains) Section Top | Title Page
James O'Connor, MS, MT (ASCP)
Tympanocentesis fluid -- Streptococcus pneumoniae Section Top | Title Page
James O'Connor, MS, MT (ASCP)
Tympanocentesis fluid -- Haemophilus influenzae Section Top | Title Page
James O'Connor, MS, MT (ASCP)
Tympanocentesis fluid -- Moraxella catarrhalis Section Top | Title Page
James O'Connor, MS, MT (ASCP)
1. Gonorrhea is a very common sexually transmitted disease seen in both males and females. Males are usually symptomatic and develop an acute urethritis. Pus from the urethra can be obtained by inserting a cotton or rayon urogenital swab 2 cm into the urethra and rotating gently before withdrawing. When profuse urethral discharge is present, the discharge may be collected without inserting a sampling device. Males who engage in homosexual activity may also develop gonococcal proctitis; anal swabs may be useful in the isolation of Neisseria gonorrhoeae. Females on the other hand tend to be asymptomatic when infected with Neisseria gonorrhoeae. Endocervical specimens are most often obtained for culture by inserting a speculum into the vaginal canal to allow visualization of vaginal and cervical architecture. After ectocervical mucus is adequately removed a urogenital dacron or cotton swab is inserted into the endocervical canal for 30 seconds and rotated before removal. Anal swabs may sometimes be useful for isolation of N. gonorrhoeae from females since these organisms may travel from the vaginal canal to the anal area.
2. Excellent recovery of gonococci is the rule when urethral,
cervical or anal swabs are immediately inoculated onto Jembec plates 
(Figure #2). The swab containing material is rolled across the
agar in a W pattern with constant turning to expose all surfaces to
the medium. The W pattern can then be cross-streaked with a sterile
inoculating loop.
3. The Jembec plate consists of modified Thayer-Martin selective medium containing hemoglobin supplement, V factors, vancomycin (inhibits gram positive organisms), colistin (inhibits gram negative rods and saprophytic neisseria), nystatin (inhibits yeasts) and trimethoprim lactate to inhibit pseudomonas and swarming proteus. A sodium bicarbonate tablet inserted into a plastic chamber in the plate generates 5% CO2 which is required by N. gonorrhoeae for growth. The plate with its plastic lid is placed in a CO2 impermeable zip-lock bag and incubated at 35 degrees C for 24-48 hours.
4. If growth occurs the plate can be sent to a reference laboratory for Gram stain, oxidase test and other biochemical studies for identification of N. gonorrhoeae.
5. In addition to culture, the urethral discharge may be examined
by Gram stain for the presence of gram-negative intracellular
diplococci (GNID) usually indicative of gonorrhea in males 
(Slide
#9). The urethral swab should be rolled over the surface of a
glass slide, covering an area the size of a nickel or quarter. If the
Gram stain results are characteristic, cultures of urethral discharge
need not be done. Urethral smears from females may also be examined,
but presumptive diagnosis of gonorrhea from vaginal smears is usually
not reliable since normal vaginal flora may contain saprophytic Neisseria sp., Veillonella sp., Moraxella osloensis and other
gram negative coccobacilli which may resemble gonococci. If the
microscopist does see extracellular organisms that resemble Neisseria gonorrhoeae, the smear should be examined for a
longer period of time for intracellular diplococci. Confirmatory
cultures or an alternate antigen detection system should always be
performed on female specimens.
James O'Connor, MS, MT (ASCP)
James O'Connor, MS, MT (ASCP)
Urethral smear with gram negative intracellular diplococci Section Top | Title Page
James O'Connor, MS, MT (ASCP)
1. Urinary tract infections (UTI) are very common infections especially in the female population. Approximately 50% of all clinical specimens received in the clinical microbiology laboratory for culture are clean-voided midstream (CVMS) urine specimens. It is of utmost importance that the genital area of the patient be properly cleansed before collecting the CVMS urine specimen. Failure to do so may result in a report of "multiple organisms present suggesting contamination." If the number of colony forming units (CFU) obtained on a properly collected CVMS urine is 50,000 CFU/ml or greater for a single potential pathogen this will indicate UTI. This count with two potential pathogens is a "probable UTI" but 3 or more organism types is a "multiple contamination" regardless of type of organism.
2. Collecting a CVMS urine on a male patient is fairly simple. The glans penis is cleansed with a gauze pad containing a soap solution. The soap is removed with a gauze pad containing sterile distilled water. The patient then voids for several seconds into the toilet and then collects a midstream specimen in a sterile container. The voiding process removes contaminating bacteria that may be present in the external portion of the urethra. The urine should be cultured immediately or placed in the refrigerator until culturing can be done.
3. There is a greater chance for contamination of urine specimens on female patients because the genital area contains many normal flora bacteria. The patient should be properly instructed on how to cleanse her genital area. This entails spreading the labia and cleansing the labia and external urethral meatus with several gauze pads soaked with soap solution, starting at the top of the labia and sweeping down. This procedure is repeated using several gauze pads soaked with sterile distilled water. This should result in removal of mucus secretions and contaminating bacteria. This patient then voids for several seconds in the toilet and collects a midstream specimen in a sterile container.
4. A quantitative culture can be easily performed using a 1:1000
ml (0.001 ml) platinum quantitative loop. The CVMS urine is first
mixed properly. The quantitative loop is sterilized in a Bunsen
burner or bacterial incinerator and then dipped vertically into the
mixed urine specimen. The loop is inoculated onto a blood agar plate
making a single line streak from the top of the plate to the bottom
of the plate 
(Figure
#3). A regular sterilized inoculating loop is used for
cross-streaking to spread the organisms over the surface of the plate
to obtain countable colonies 
(Figure
#4). The same procedure can be done on an Eosin-methylene blue
(EMB) plate which is a selective and differential medium for gram
negative rods. The plates are incubated overnight at 35 degrees C and
examined the next day for countable colonies. The number of colonies
present multiplied by 1000 gives the number of organisms/ml of urine.
5. The most common cause of UTI is E. coli, a gram negative rod. The colonies on blood agar are large (3-5 mm), gray and shiny and have a distinct "wet wool" odor suggestive of moth balls. They may be beta or non-hemolytic. On EMB agar the colonies are somewhat flat, opaque purple colonies with a target appearance indicating lactose fermentation. They usually have a green metallic sheen.
6. The second most common cause of UTI are the gram negative rods belonging to the Klebsiella-Enterobacter group. These organisms are usually encapsulated and the colonies on blood agar are large, gray, shiny and quite mucoid. They have a "horse barn" or "livestock" odor. On EMB they are quite mucoid with a purple opaque target appearance.
7. Proteus sp. are probably the third most common cause of UTI. These organisms are easily identified by their ability to spread or swarm over the entire surface of the blood agar and EMB plates due to their highly motile characteristics. They exhibit a faint odor of chocolate or brownie mix. If plate is older a strong ammonia smell may occur.
8. Staphylococcus and streptococcus colonies are much smaller than gram negative rod colonies and lack the "stinky" gram negative rod odor. The gram stain will show gram positive cocci in grape-like clusters for staphylococci. Streptococci will be gram positive cocci in singles, pairs and short chains. The cocci may also appear somewhat elongated. Staphylococci and streptococci do not grow on EMB. The staphylococci can be easily differentiated from streptococci by the catalase test. When a colony of a staphylococcus is placed in a drop of 3% hydrogen peroxide on a microscope slide, foaming or bubbling will occur (positive test). Streptococci are catalase negative (no foaming or bubbling). Staphylococcus aureus can be differentiated from other Staphylococcus sp. by the slide or tube coagulase test. The slide coagulase test is performed by mixing a colony of the staphylococcus in a drop of rabbit plasma on a microscope slide. If the organism is S. aureus, clumping of the mixture occurs due to the production of clumping factor (positive slide coagulase test). The other Staphylococcus sp. are slide coagulase test negative and the suspension of organisms mixed with plasma remains smooth or homogeneous. In the tube coagulase test the organism is mixed with 0.5 ml of rabbit plasma in a test tube followed by incubation at 35 degrees C for 2-4 hours. S. aureus produces coagulase enzyme which converts the fibrinogen in plasma to fibrin resulting in clot formation indicating a positive tube coagulase test. Other Staphylococcus sp. give a negative tube coagulase test and the plasma remains liquid. There are also latex kits (Staph aurex, Serostat Staph etc.) on the market that can be easily used for identification of Staphylococcus aureus. The reagent consists of latex particles coated with plasma (contains fibrinogen and IgG). A colony of the S. aureus is mixed with a drop of latex reagent on a slide. Clumping factor in the cell wall of S. aureus converts fibrinogen to fibrin resulting in clumping of the latex particles and Staphylococcus aureus. S. aureus also contain protein A in the cell wall which binds to the Fc region of IgG on the latex particles. This also results in clumping of the S. aureus cells with the latex particles. Other staphylococci give a negative test (no clumping of the plasma coated latex particles) because they lack clumping factor and protein A.
9. Plastic dip-slides may also be used for quantitating bacteria
in urine. These are covered on both sides with various culture media.
The dip-slide is affixed to a plastic cap and the slide/cap
combination is contained in a sterile plastic vial 
(Figure
#5). The dip-slide is dipped into a properly mixed CVMS
urine specimen. If the urine contains bacteria, some of these will
adhere to the surfaces of the media. The number of bacteria on the
media surfaces is proportional to the bacterial concentration in the
urine specimen. The slide is placed in a sterile plastic vial and
incubated at 35 degrees C for 18-24 hours. Bacterial colonies form on
the agar surface and the bacterial count of the urine specimen is
determined by comparing the colony density on the slide with a model
chart provided with the dip-slide kit. One can also purchase
"dip-slides" with a variety of media to assist in isolation and
identification of the pathogen responsible for the UTI.
James O'Connor, MS, MT (ASCP)
James O'Connor, MS, MT (ASCP)
James O'Connor, MS, MT (ASCP)
James O'Connor, MS, MT (ASCP)
1. Enterobiasis (pinworm disease) is caused by the nematode worm Enterobius vermicularis. This organism has a worldwide distribution and can infect horses, mice and primates (man and monkeys). It does not infect cats and dogs. Enterobiasis is the most common human helminth disease in the U.S.A. Most infections occur in children.
2. Humans become infected by ingestion of infected embryonated eggs which reach the mouth by inhalation, hand to mouth transport, or in contaminated food or drink. Infections many times involve families living under crowded conditions. Sometimes the entire family may be infected.
3. Male and female adult worms live in the colon and mate. When the female becomes gravid (filled with eggs) she migrates through the anus (usually at night) to the perianal region where she lays sticky eggs over the perianal skin. Severe perianal itching (pruritus) due to the female worm's uterine fluid can cause loss of sleep and chronic hyperirritability in a child. Scratching of the anal area may lead to dermatitis and secondary bacterial infections. Scratching also dislodges the eggs providing a potential source of infection to sibling and parents.
4. Diagnosis is accomplished by the scotch tape (cellulose tape)
method 
(Figure
#6). The sticky part of the clear transparent cellulose tape is
applied to the perianal region usually in the morning when the
patient first awakes. Eggs stick to the transparent tape. The tape is
placed sticky side down on a glass microscope slide and examined
microscopically for the very characteristic eggs which are 55 um x 25
um wide, ovoid with a thick clear shell and flattened on one
side. Internally there is a developing larva. See 
Figure
#7 for the typical appearance of Enterobius vermicularis eggs. 
Slide
#10 shows the microscopic appearance of a positive scotch tape
preparation. Sometimes the adult female worm may even be present in
the scotch tape preparation. The female is a spindle-shaped worm
about 1/2 inch long and 0.5 mm wide. The anterior end has
characteristic structures called dorsoventral cephalic inflations or alae. The esophagus terminates in a distinct esophageal bulb.
The posterior end is long, pointed (hence "pinworm") and perfectly
clear (devoid of any internal structures). The uteri are distended in
the gravid female so that the entire body is packed with eggs except
for the clear pointed tail. See 
Figure
#8 for a drawing of the adult female worm. There is a
commercially available plastic "paddle" with a flat sticky surface
that can be used to collect the specimens. Sometimes problems with
clarity of the preparation may occur and a drop of toluene added to
the preparation may alleviate the problem. The pinworm eggs are
infectious even on the scotch tape or slide, so handle carefully.
James O'Connor, MS, MT (ASCP)
James O'Connor, MS, MT (ASCP)
James O'Connor, MS, MT (ASCP)
Enterobius ova (Scotch tape prep) Section Top | Title Page
James O'Connor, MS, MT (ASCP)
James O'Connor, MS, MT (ASCP)
1. The brochure accompanying the kit should be read carefully before performing the test. It will give an explanation of the principle and summary of the test, preparation of reagents, storage instructions, specimen collection and handling and interfering substances.
2. The RPR card test is a non-treponemal test for the serological detection of syphilis. Either clear unhemolyzed serum or plasma from EDTA anticoagulated blood may be tested. The antigen suspension consists of carbon particles and a cardiolipin-lecithin extract of beef heart. This non-specific antigen detects reagin, an IgM or IgG immunoglobulin present in the plasma or serum of patients with syphilis and occasionally present in the serum or plasma of individuals with other acute or chronic conditions. If reagin is present it binds to the cardiolipin antigen resulting in flocculation. The carbon particles become trapped in the flocculation and appear agglutinated or as black clumps against a white background on the test card. The agglutination can be seen macroscopically. Specimens that are nonreactive, that is, do not contain reagin, have a uniform light-gray color and even particle distribution (no clumping).
3. The antigen suspension is mixed thoroughly and taken up into a small plastic dispensing vial containing a needle. The needle should be previously checked for dispensing accuracy by placing it firmly on a 1 ml pipet filled with the RPR antigen suspension. The pipet is held vertically and the number of drops delivered in 0.5 ml. is counted. The correct number of drops is 30 + or - 1 drop.
4. The 18 mm qualitative card test is first performed on the
patients specimen using special pipet/stirrers 
(Figure #9). The pipet/stirrer is like a straw--it is open on one
end and has a flat paddle or flap on the opposite end. The
pipet/stirrer is squeezed and the open end inserted into the
specimen. Releasing pressure will draw sample up into pipet. One free
falling drop (50 ul) is dispensed into an appropriate circle on the
test card 
(Figure #10). The flattened paddle end of the pipet/stirrer is
used to spread the mixture over the entire area of the circle. The
antigen dispensing bottle is then mixed and held in a vertical
position, dispensing several drops into the cap to make sure the
needle passage is clear. One free falling drop is then dispensed into
the appropriate circles. Stirring should not be done since mixing of
the antigen suspension and serum will occur during rotation. The test
card is immediately placed on a mechanical rotator, a humidifier
cover applied and rotated at 100 RPM for 8 minutes. Following
rotation a brief rotating and tilting of the card by hand (3-4 to and
fro motions) must be made to aid in differentiating non-reactive from
minimally reactive results. The card is immediately read
macroscopically in the wet state under a high intensity incandescent
lamp.
5. Reporting of results
6. Quantitative RPR Test
7. Quality Control
8. The RPR test will be positive in patients who have venereal syphilis and other treponemal diseases, such as bejel, pinta and yaws. Biological false positive (BFP) reactions may occur with cardiolipin antigen suspensions due to the presence of substances which react like reagin in the serum of persons having a variety of acute or chronic infections. BFP's may be caused by such conditions as infectious mononucleosis, pregnancy, bacterial and viral infections, SLE, myeloma, rheumatoid arthritis, lymphoma, leprosy, atypical pneumonia, malaria, etc.
9. The RPR test has high sensitivity (positive in disease) since approximately 80% of people with primary syphilis and 99% with secondary syphilis will give a positive test. It is therefore an excellent screening test. It is also easy to perform, fast and inexpensive. The test does, however, have low specificity and many other conditions as mentioned previously may give biological false positives.
James O'Connor, MS, MT (ASCP)
James O'Connor, MS, MT (ASCP)
Section Top | Title Page