MICROBIOLOGY - TABLE OF CONTENTS

• Microbiology Test Comments
• Instructions for Completing RML Test Request II
• Time Required for Culture Results
• Quantitation of Microbiology Smears and Cultures
• Specimen Collection and Transport System (Transwab)
• Specimen Requirements by Culture Source
• Aerobic Culture Collection Procedure (Routine)
• Anaerobic Culture Collection Procedure
• Blood Culture Collection Procedure (Venous)
• Chlamydia/GC RNA Probe Collection Procedure
• Clostridium Difficile Cytotoxin Collection Procedure
• Direct Fungal Prep/Smear Collection (Replaces KOH)
• Fungus Culture Collection Procedure
• Genital Culture Collection Procedure
• Herpes virus Culture Collection Procedure
• Influenza A/B Antigen Specimen Collection
• Nasal and Throat Washing, Aspiration or Nasal Pharyngeal Swab Collection
• Ova and Parasites Collection Procedure
• Pinworm (Enterobius vermicularis) Collection Procedure
• Rotavirus Detection Collection Procedure
• Sputum Culture Collection Procedure
• Stool Culture/Rectal Swab Collection Procedure
• Throat Culture Collection Procedure
• Urine Culture Collection Procedure - Clean Voided Midstream
• Urine Culture Collection Procedure - Indwelling Catheter
• Wet Prep for Trichomonas

Please contact our Microbiology Department for any questions concerning Microbiology or Infection Control.

MICROBIOLOGY TEST COMMENTS

CONTENTS:

• Susceptibility Report Key
• Empiric Therapy Guidelines
• Organism Specific Comments

SUSCEPTIBILITY KEY:

• Blac = Beta-lactamase Pos (B-lactamase antibiotic resistant)
• EBL? = Suspected ESBL
• N/R = Not advised or tested
• ESBL = Extended Spectrum Beta-lactamase
• R = Resistant
• R* = Resistant, ESBL
• I = Intermediate
• S = Susceptible
• TFG = Thymidine-dependent strain (sulfa resistant)

Amox/K Clav; Amp/Sulbactam — For Streptococci (including Enterococci), Micrococci and Listeria species, refer to ampicillin interpretation. If ampicillin results are unavailable, refer to penicillin. If penicillin results are resistant, test ampicillin using an alternative method.

Blank Interpretation Column — Drug not advisable or no activity against this organism.

Cefuroxime — Breakpoints based upon parenteral doses. For cefuroxime PO: <8 = S, 8-16 = I, 16 = R.

EBL? (suspected ESBL) indicates an ESBL is suspected but has not been confirmed; the organism will be held for four days in the event that confirmation is required. Contact the Microbiology Department for further testing.

ESBL (Extended Spectrum Beta-lactamases) are plasmid-mediated Beta-lactamases which confer resistance to first and third generation cephalosporins but not to the cephamycins (second generation cephalosporins). A typical antibiogram would be resistant to cephalothin, ceftazidime and aztreonam, while susceptible to cefoxitin and cefotetan. Organisms should remain susceptible to carbapenems and combination drugs such as ticarcillin/K clavulanate. While usually associated in the U.S. with Klebsiella pneumoniae, they are less commonly seen with E. coli, Serratia marcescens, Citrobacter freundii and Enterobacter cloacae. It appears that these enzymes are selected by widespread use of third generation cephalosporins and aztreonam. It is possible for organisms to acquire and express multiple Beta-lactamase enzymes, making treatment a dilemma.

IB appears in place of S or I with species known to possess inducible B-lactamases; potentially they may become resistant to all B-lactam drugs such as penicillin, ampicillin, cephalosporins and cephamycins, and ultimately monobactams (aztreonam). Plasmid-mediated Beta-lactamases are expressed constitutively, i.e., constantly, and are thus easily detected as the organism always appears resistant. These plasmid-mediated enzymes are susceptible to inhibition by Beta-lactamase inhibitors such as clavulanic acid and sulbactam in combination with ampicillin or amoxicillin. A large number of Enterobacteriaceae and Pseudomonaceae produce Beta-lactamases by the effect of a gene located on the bacterial chromosome. These enzymes are only expressed after exposure of the organism to Beta-lactam antibiotics. Inducible Beta-lactamase enzymes are not susceptible to inhibition by clavulanate or sulbactam. Physicians who may wish to avoid use of Beta-lactam antibiotics in treating infections with organisms which have inducible Beta-lactamases; if Beta-lactam antibiotics are used, it is recommended that the patient be monitored with repeated cultures during and after therapy. Avoid other/combined Beta-lactam drugs.

R* appears with Klebsiella spp. and E. coli with increased resistance (>2 mcg/ml) MICs to cefpodoxime, ceftazidime or aztreonam; these may harbor an ESBL (Extended Spectrum Beta-lactamase) which would make them clinically resistant to all cephems and aztreonam. Cefotaxime and ceftriaxone may also detect ESBLs but are less sensitive.

Ticar/K Clav — Use maximum doses of drug with an aminoglycoside for Pseudomonas aeruginosa in patients with granulocytopenia or serous infections.

Thymidine dependent strains have developed an alternative biochemical pathway to avoid the action of sulfa-containing antibiotics. These bacteria will have an MIC value which suggests susceptibility to sulfa antibiotics in vivo. However, use may result in treatment failure as the organism will use thymidine from the patient cells to circumvent the debilitating effect of the antibiotic.

EMPIRIC THERAPY GUIDELINES: (Unless otherwise noted, based upon Mandell. Principles and Practice of Infectious Diseases: Antimicrobial Therapy, 1993/1994.)

Anaerobic Empiric Therapy — Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Peptostreptococcus and Clostridium spp.: Amp/sulbactam, imipenem, chloramphenicol >95% ticar/clavulanate, metronidazole = 85-95%. Bacteroides gracilis: Imipenem, chloramphenicol >95%, metronidazole = 85-95%.

Anaerobic Non-Spore Forming Gram-Positive Bacilli Empiric Therapy (e.g., Propionibacterium, Lactobacillus, Eubacterium, Actinomyces spp.) — Penicillin, imipenem, chloramphenicol, amp/sulbactam, ticar/clavulanate, cefotaxime, ceftizoxime >95%.

Group A, C and G Streptococci show predictable response to empiric therapy with penicillin, erythromycin, cephalosporins, vancomycin, clarithromycin, azithromycin and clindamycin. No susceptibility performed according to NCCLS guidelines.

Group B Streptococci show predictable response to empiric therapy with penicillin, ampicillin (with or without aminoglycocides), cephalosporins and vancomycin. Susceptibility testing may be indicated if patient is allergic to penicillin.

Beta Streptococci, not Groups A, B, C OR G generally show predictable response to empiric therapy with penicillin, ampicillin, erythromycin, clindamycin and vancomycin. (Gorbach, 1992.)

Enterococcus isolates from serious systemic infections such as endocarditis require combination therapy of penicillin or ampicillin, ampicillin/sulbactam or vancomycin together with an aminoglycoside (gentamicin, amikacin or streptomycin) or teicoplanin alone. Testing to confirm appropriateness of this therapy for this isolate will be performed.

Haemophilis Influenzae isolates are not routinely tested for susceptibility to ampicillin/clavulanate due to technical limitations. Isolates which are ampicillin-susceptible should be considered susceptible to this agent, as should ampicillin-resistant strains which are Beta-lactamase-positive. Isolates which are Beta-lactamase-negative and ampicillin-resistant may be resistant to Beta-lactamase inhibitor combination drugs due to non-Beta-lactamase mediated mechanisms but the occurrence of this type of resistance is very low (<1% of RML experience).

Moraxella (Branhamella) Catarrhalis generally shows predictable response to empiric therapy with trimethoprim-sulfamethoxazole, erythromycin and other macrolides, amoxicillin-clavulanate, tetracycline and various second and third generation cephalosporins. No susceptibility performed according to NCCLS guidelines.

Neisseria Meningitidis generally shows predictable response to empiric therapy with penicillin, cefotaxime, ceftizoxime, ceftriaxone, chloramphenicol and sulfonamides. No susceptibility performed according to NCCLS guidelines.

Non-Enterococcal Alpha AND Gamma Streptococci generally show predictable response to empiric therapy with penicillin (with or without gentamicin), cephalosporins and vancomycin. No susceptibility performed according to NCCLS guidelines.

Streptococcus Pneumoniae isolate screened for resistance; expect predictable response to empiric therapy with penicillin, cephalosporins, vancomycin (with or without rifampin), trimethoprim-sulfamethoxazole, macrolides, clindamycin and chloramphenicol, according to the Medical Letter. No susceptibility performed according to NCCLS guidelines.

ORGANISM SPECIFIC COMMENTS:

Corynebacterium Jeikeium can be found as part of the normal flora of the skin, particularly the inguinal, perirectal, axillary and inter-triginous areas. Colonization occurs most commonly among the immune compromised, especially those who have received multiple courses of antibiotics. C.jeikeium has been associated with sepsis, prosthetic valve endocarditis, urinary tract infections, pyelonephritis, peritonitis, meningitis and cavitating pneumonia. The organism is characteristically resistant to multiple antibiotics. Vancomycin is the drug of choice in serious infections; in less serious infections, tetracycline or erythromycin may be effective. (MCM VI)

Corynebacterium Minutissimum is the etiologic agent of erythrasma. Although generally a superficial infection, recurrent deep tissue breast abscess has been noted in apparently healthy individuals. Septicemia and graft infections have also been recorded. If susceptible by screening method, the drugs of choice are penicillin or erythromycin. (MCM VI)

Corynebacterium Striatum has been found as part of the normal flora of the human nasal passages. C.striatumhas been associated with pneumonia and lung abscess in immuno-competent individuals, as well as those with COPD. If susceptible by screening method, the drugs of choice are penicillin and erythromycin. (MCM VI)

Corynebacterium Ureolyticum (formerly CDC GRP D-2) is found on the skin as a saprophyte. It has been associated with chronic UTI and alkaline-encrusted cystitis, as well as pyelonephritis and osteomyelitis. It has also been recovered from blood cultures in cases where UTI has persisted. The organism has a propensity for stone formation due to presence of a strong urease enzyme. Suggested empiric therapy includes norfloxacin and vancomycin as the agent is commonly resistant to Beta-lactam, aminoglycoside and macrolide antimicrobial agents. Fluoroquinolones show irregular activity. (Janda & MCM VI)

Non-Pathogenic Parasites — Entamoeba hartmanni, Entamoeba coli, Entamoeba polecki, Endolimax nana, Iodamoeba butschlii and Chilomastix mesnili are non-pathogenic intestinal protozoan parasites which generally produce no evidence of disease. Presence of these organisms, however, does indicate exposure to fecal contamination; complete testing for bacterial, viral and other intestinal parasitic agents should be considered.

Parasites of Indeterminate Pathogenicity — Previously thought to be an amoeba, Blastocystis hominis has recently been identified as belonging to a diverse group including some algae and diatoms. It inhabits the large intestine and may be found in up to 25% of stools; only occasional patients have clinical symptoms. Blastocystitis may be suspected to be pathogenic in a patient when testing for bacterial, viral and other parasitic agents is negative AND blactocystitis is numerous and persistent. The symptom generally associated is persistent, mild diarrhea.

Pathogenic Parasites — Entamoeba histolytica, Dientamoeba fragilis and Giardia lamblia are pathogenic protozoan parasites. These organisms may not produce symptoms in infected patients or may remain after symptoms have resolved, thus providing the potential for transmission to others. In addition, presence of one of these agents does not exclude the presence of another fecal pathogen such as salmonella, shigella or rotavirus. Dientamaoba is noted to be associated with the presence of enterobius (pinworms). All of these agents are reportable to the local health department.

Salmonella isolates are tested for their susceptibility to antibiotics to rule out the presence of DT104 strain which conveys multi resistance (to ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline), as well as increased virulence. Uncomplicated gastroenteritis associated with salmonella and most other bacterial agents is usually self-limiting and resolves without treatment; antimotility agents may prolong exposure to toxins and antibiotic treatment may prolong carriage and increase resistance.

INSTRUCTIONS FOR COMPLETING RML TEST REQUEST II FOR MICROBIOLOGY SPECIMEN

1. Patient Information — Please complete the Test Request II form as stated in the "Requisition" section.
2. Type of Culture — This refers to the type of work-up requested. PLEASE give diagnosis # or code to the left of the type of culture ordered (i.e., aerobic with smear, fungus, etc.).
3. Source of Culture — This refers to the source of the culture material. Please give exact anatomical location where culture was obtained (example: left leg, right index finger, ear, etc.). PLEASE NOTE: Separate requisitions are required when submitting blood work collected at different times, ie., blood cultures and other blood work. Submit SEPARATE requisitions when sending culture samples FROM DIFFERENT SOURCES.
4. Sensitivity — Please check if needed. If a pathogen is recovered and no susceptibility has been requested, the report will indicate that you have four days to request the sensitivity.
5. Diagnosis — Be sure to indicate the appropriate diagnosis for the culture submitted.
6. Comment — List any additional information which may be helpful regarding the culture (i.e., recurrent infection, antibiotic therapy used prior to culture) in the "Comment" section.

MICROBIOLOGY DEPARTMENT
NORMAL TIME REQUIRED FOR ROUTINE CULTURE RESULTS

• Acid Fast (TB)
  • Preliminary Report: 48 hours and 4 weeks
  • Final Report: Incubated 7 weeks
• Anaerobes
  • Preliminary Report: Gram stain* at 24 hours; preliminary report at 48 hours
  • Final Report: Incubated 7 days
• Blood Cultures
  • Preliminary Report: 48 hours
  • Final Report: Incubated 7 days
• Body Fluids
  • Preliminary Report: Gram stain* and preliminary report at 24 hours
  • Final Report: Incubated 7 days
• Eye, Ear
  • Preliminary Report: Gram stain* and preliminary report at 24 hours
  • Final Report: 48 to 72 hours
• Fungi
  • Preliminary Report: 1 week
  • Final Report: Incubated 4 weeks
• GC
  • Preliminary Report: Gram stain* (males only) and preliminary POSITIVE at 24 hours
  • Final Report: 24 hours (M-F only)
• Genital
  • Preliminary Report: Gram stain* and preliminary POSITIVE if significant isolates at 24 hours
  • Final Report: 72 hours
• Influenza A/B Antigen
  • Final Report: Same day if in laboratory by 3:00 PM
• Sensitivities
  • Final Report: Generally 48-72 hours after culture received
• Spinal Fluid
  • Preliminary Report: Gram stain STAT if requested (otherwise, preliminary report at 24 hours)
  • Final Report: Incubated 7 days
• Sputum
  • Preliminary Report: Gram stain* and preliminary report at 24 hours
  • Final Report: 48 hours
• Strep Screen (Rapid)
  • Final Report: Same day if in laboratory by 2:30 PM
• Stool
  • Final Report: 72 hours
• Throat Culture
  • Preliminary Report: If positive, preliminary report at 24 hours
  • Final Report: 48 hours
• Urine
  • Preliminary Report: 24 hours
  • Final Report: 48 hours
• Wound, Ulcer, Abscess
  • Preliminary Report: Gram stain* and preliminary report at 24 hours
  • Final Report: 7 days

* For Gram stain interpretation, refer to "Quantitation of Microbiology Smears and Cultures"

QUANTITATION OF MICROBIOLOGY SMEARS AND CULTURES

To assist physicians in assessing the clinical significance of reports from the Microbiology laboratory, we will use a system of quantitation of direct smears of specimens and of bacterial plate growth.

GRAM STAIN REPORT — Direct Gram stains of wounds, sputums, exudates, spinal fluids and genital cultures are performed at no additional cost. We will report these smears generally the same day the specimen is received, using the criteria shown in Table 1. The term element refers to WBCs and epithelial cells, as well as to bacterial cells.

TABLE 1

REPORT FINDING ON GRAM STAIN

4+ >30 elements/oil immersion field (1,000X)
3+ >5-30 elements/oil immersion field
2+ >1-5 elements/oil immersion field
1+ 1 element/oil immersion field
Few 1 element/5 oil immersion fields
Rare <1 element/5 oil immersion fields

CULTURE REPORT — Listing organisms in order of predominance of their growth in a culture is not sufficient information to the physician. We will quantitate the growth on all cultures except urines by the schema presented in Table 2, according to the amount of growth of each organism in the three streak areas of the plate. Normal flora will also be graded in this fashion. Using this criteria, heavy growth of an organism would be represented by a value of 3+ or greater.

TABLE 2 — NUMBER OF COLONIES IN STREAK AREA

GRADE 1ST AREA 2ND AREA 3RD AREA

Few 0-5

1+ <10

2+ >10 <5

3+ >10 >5 <5

4+ >10 >5 >5

Peel open envelop to expose caps.

1. Remove cap from transport medium tube and discard it.
2. Remove swab from package, holding by cap only. Collect the specimen
3. Place swab in transport medium, capping firmly.
4. Label transport tube itself with patient's identification sticker from Test Request.

SPECIMEN REQUIREMENTS BY CULTURE SOURCE

ABSCESS, WOUND OR EXUDATES:

• AEROBIC (Routine)
  • Equipment Needed:
    • Sterile swab transport unit
    • Sterile saline, if necessary
    • Sterile cotton gauze, if needed
    • Sterile scalpel blade, if needed
  • Specimen Collection:
    • See procedure in MICRO section. Aseptically open wound if not draining. Swab deepest portion of lesion, touching only sterile cotton tip of swab to site. Place the swab, cotton tip first, into the transport medium, capping securely.
  • Holding and Transport:
    • Room temperature. DO NOT REFRIGERATE.
• ANAEROBIC
  • Equipment Needed:
    • ACT I tube
    • Sterile syringe and needle for aspiration
    • Extra sterile needle
    • Iodophor swab
    • Alcohol pad
    • Sterile cotton swab if unable to aspirate
    • Sterile saline, if needed
    • Sterile cotton gauze, if needed
  • Specimen Collection:
    • See procedure in MICRO section. Aseptically collect specimen with syringe. Replace needle with fresh sterile needle and inject into ACT I tube. If collected with swab, place cotton tip of swab deep into ACT I tube and break off wooden stick. Replace cap of transport medium.
  • Holding and Transport:
    • Room temperature. DO NOT REFRIGERATE.

EAR (EXTERNAL CANAL), ROUTINE:

• Equipment Needed:
  • Sterile swab transport unit
• Specimen Collection:
  • See procedure in MICRO section. Swab external auditory canal with sterile cotton swab. Place cotton tip deep into transport medium, capping securely.
• Holding and Transport:
  • Room temperature.

EYE, ROUTINE:

• Equipment Needed:
  • Sterile swab transport
• Specimen Collection:
  • BEFORE TOPICAL ANTIBIOTICS ARE USED, wipe any white matter which has accumulated in the eye and the internal surface of the lower lid with swab. Place the fiber?covered end of the swab in the transport medium, capping securely.
• Holding and Transport:
  • Room temperature. DO NOT REFRIGERATE.

CSF FLUIDS, ROUTINE:

• Equipment Needed:
  • Sterile tube #3 from LP pack
• Specimen Collection:
  • 2.0 cc CSF minimum.
• Holding and Transport:
  • Room temperature. DO NOT REFRIGERATE.

BLOOD, AEROBIC AND ANAEROBIC:

• Equipment Needed:
  • Aerobic and anaerobic BacT/Alert bottles
  • Sterile 20 cc syringe with 22 gauge needle
• Specimen Collection:
  • See procedure in MICRO section. BEFORE ADMINISTRATION OF ANTIBIOTICS, aseptically collect 20 cc of venous blood. Wipe rubber stopper of each bottle with iodine and inject 10 cc into each bottle.
• Holding and Transport:
• Room temperature or incubate at 35°C if available. DO NOT REFRIGERATE

ABDOMINAL, DIALYSIS, CHEST, PERICARDIAL, PERITONEAL FLUID, ROUTINE AFB, TB, FUNGUS:

• AEROBIC
  • Equipment Needed:
    • Sterile container
    • Sterile syringe with 22 gauge needle
    • Extra sterile needle
    • Iodophor swab
    • Alcohol pad
  • Specimen Collection:
    • If site is not open, wipe skin with Iodophor, then alcohol. Aspirate fluid aseptically. Change needle and aseptically transfer fluid to sterile container.
  • Holding and Transport:
    • Room temperature.
• ANAEROBIC
  • Equipment Needed:
    • A.C.T. I anaerobic transport tube
    • Alcohol pads (2)
    • Sterile syringe with 22 gauge needle
    • Iodophor swab
    • Extra sterile needle
  • Specimen Collection:
    • See procedure in MICRO section. Aspirate fluid aseptically. Wipe rubber top of transport vial with alcohol. Replace needle used to aspirate with sterile needle and inject fluid into transport vial.
  • Holding and Transport:
    • Room temperature.

GENITAL:

• Genital, Male (includes GC)
  • Equipment Needed:
    • Sterile nasopharyngeal (N/P) Transwab unit
  • Specimen Collection:
    • See procedure in MICRO section. Aseptically collect specimen from distal urethra. Place fiber end of swab into transport medium, capping securely.
  • Holding and Transport:
    • Room temperature. DO NOT REFRIGERATE.
• Genital, Female (includes GC)
  • Equipment Needed:
  • • Sterile swab transport unit
  • Specimen Collection:
    • See procedure in MICRO section. Aseptically collect specimen from distal urethra. Place fiber end of swab into transport medium, capping securely.
  • Holding and Transport:
    • Room temperature. DO NOT REFRIGERATE.
• GC Screen, Male (GC only)
  • Equipment Needed:
    • Sterile nasopharyngeal (N/P) Transwab unit
  • Specimen Collection:
    • See procedure in MICRO section. Aseptically collect specimen from distal urethra. Place fiber end of swab into transport medium, capping securely.
  • Holding and Transport:
    • Room temperature. DO NOT REFRIGERATE.
• GC Screen, Female (GC only)
  • Equipment Needed:
    • Sterile swab transport unit
  • Specimen Collection:
    • See procedure in MICRO section. Aseptically collect specimen from distal urethra. Place fiber end of swab into transport medium, capping securely.
  • Holding and Transport:
    • Room temperature. DO NOT REFRIGERATE.

NASAL:

• NOSE
  • Equipment Needed:
    • Sterile culturette swab
  • Specimen Collection:
    • Immobilize patient's head. Gently pass swab into the nostril and gently rotate. Place the fiber tip end of the swab into transport medium, capping securely.
  • Holding and Transport:
    • Room temperature.
• Nasopharyngeal Swab Collection
  • Equipment Needed:
    • Gloves
    • Mask
    • Dacron or Rayon nasopharyngeal swab. (Do not use calcium alginate or swab with wooden shaft).
  • Specimen Collection:
    • Wear gloves. A mask is recommended since this procedure may induce sneezing.
    • Explain procedure to patient or parent. If patient is a child, request assistance in stabilizing head.
    • Tip patients's head back and secure.
    • Introduce the NP swab into the nostril along the floor of the nasal cavity and near the septum until the pharyngeal wall is reached. If any obstruction is encountered, switch to the other side.
    • Rotate the swab 3 times and remove it with a rotating motion.
    • Return swab to the paper wrapper, label and refrigerate.
    • Complete the test request with patient information.
• Influenza A/B Antigen Specimen Collection Secretions of the nasopharynx are superior for the detection of Influenza and can be submitted on a nasoharyngeal swab or as an aspirate. A N/P swab submitted in 1.0 ml of saline is acceptable.
  • Equipment Needed:
    • Use dacron or rayon N/P swab in paper wrapper.
• Respiratory Syncytial Virus (RSV) Nasopharyngeal swab in paper wrapper or in 1.0 ml. saline. N/P aspirates are also acceptable.
  • Equipment Needed:
    • Use dacron or rayon N/P swab in paper wrapper.
  • Specimen Collection:
    • See Nasopharyngeal swab collection.
  • Holding and transport
    • Refrigerate. Transport to laboratory within 24 hrs.
• PERTUSSIS
  • Equipment Needed:
    • Use B. pertussis media with swab
  • Specimen Collection:
    • See Nasopharyngeal Swab Collection procedure, but make sure to use B. pertussis media transporter with swab provided.
  • Holding and Transport:
    • Refrigerate

SPUTUM:

• RESPIRATORY
  • Equipment Needed:
    • Sterile container
  • Specimen Collection:
    • See Sputum Collection procedure. First morning DEEP COUGH specimen, not saliva. 3 cc minimum.
  • Holding and Transport:
    • Refrigerate for 12 hours maximum. Transport on ice.
• AFB, TB
  • Equipment Needed:
    • Sterile container
  • Specimen Collection:
    • See Sputum Collection procedure. First morning DEEP COUGH specimen, not saliva. 3 cc minimum.
  • Holding and Transport:
    • Refrigerate for 12 hours maximum. Transport on ice.
• FUNGUS
  • Equipment Needed:
  • Sterile container
  • Specimen Collection:
    • See Fungus Collection procedure. First morning DEEP COUGH specimen, not saliva. 3 cc minimum. If histoplasmosis is suspected, contact Microbiologist at RML.
  • Holding and Transport:
    • Refrigerate for 12 hours maximum. Transport on ice.

STOOL OR RECTAL SWAB: (NOTE: Stool specimens for culture must be obtained either prior to barium administration [as for x-rays] or delayed a minimum of TEN DAYS after its administration)

• STOOL
  • Equipment Needed:
    • Sterile container or stool transport or TWO sterile swab transport units
  • Specimen Collection:
    • See Stool Culture Collection procedure. Collect stool specimen or TWO rectal swabs. Place stool specimen in sterile container (or stool transport). Place swabs in stool transport and cap securely.
  • Holding and Transport:
    • If STOOL specimen, refrigerate and transport within one hour. IF IN TRANSPORT, can hold at room temperature and transport within 24 hours.
• ROTAVIRUS
  • Equipment Needed:
    • Sterile urine container
  • Specimen Collection:
    • See Rotavirus Collection procedure. 2 cc stool required.
  • Holding and Transport:
    • Refrigerate and Transport on ice.

THROAT:

• ROUTINE
  • Equipment Needed:
    • Sterile swab transport unit
  • Specimen Collection:
    • See Throat Culture Collection procedure. Swab posterior pharynx. Place cotton tip end of swab deep into transport medium, capping securely.
  • Holding and Transport:
    • Room temperature.
• STREP ANTIGEN ONLY
  • Equipment Needed:
    • Sterile swab transport unit
  • Specimen Collection:
    • See Throat Culture Collection procedure. Swab posterior pharynx. Place cotton tip end of swab deep into transport medium, capping securely.
  • Holding and Transport:
    • Room temperature. NOTE: Specimen must be received by 2:30 PM for results by 4:30 PM the SAME DAY.
• GC
  • Equipment Needed:
    • Sterile swab transport unit
  • Specimen Collection:
    • See Genital Culture Collection procedure. Swab posterior pharynx. Place cotton tip end of swab deep into transport medium, capping securely.
  • Holding and Transport:
    • Room temperature.

URINE:

• ROUTINE (Clean Voided)
  • Equipment Needed:
    • Sterile urine container
    • Sterile cotton balls or gauze; sterile sponges
    • Green soap cleansing solution
    • Sterile water to rinse off cleansing solution
    • Disposable gloves, if needed
  • Specimen Collection:
    • See Urine Culture Collection procedure. Collect 10 cc of urine. First morning specimen is best.
  • Holding and Transport:
    • Refrigerate (maximum 18 hours). Transport on ice.
• CATHETERIZED
  • Equipment Needed:
    • Sterile 12 cc syringe with 22 gauge needle
    • Alcohol sponge
    • Clamp or rubber band
    • Sterile urine container
  • Specimen Collection:
    • See Urine Culture Collection procedure. Collect 10 cc of urine. Not necessary to have first morning specimen.
  • Holding and Transport:
    • Refrigerate (maximum 18 hours). Transport on ice.
• AFB (TB)
  • Equipment Needed:
    • Sterile urine container
    • Sterile cotton balls or gauze; sterile sponges
    • Green soap cleansing solution
    • Sterile water to rinse off cleansing solution
    • Disposable gloves, if needed
    • NOTE: Do not recommend catheterization solely to collect urine for AFB culture)
  • Specimen Collection:
    • See Urine Culture collection procedure. Collect entire first morning specimen. DO NOT submit 24 hour collection.
  • Holding and Transport:
    • Refrigerate (maximum 18 hours). Transport on ice.

AEROBIC CULTURE COLLECTION PROCEDURE (ROUTINE)

For material needed, consult guideline in MICRO under specific type of specimen (i.e., wound, eye).

PROCEDURE:

BE SURE THE STERILE SWAB TRANSPORT IS AT ROOM TEMPERATURE.

1. If source is a lesion
   • Remove dressing, if present, from area to be cultured
   • Wash area gently with sterile saline and sterile cotton gauze to remove encrusted material, if present.
   • Open lesion carefully with sterile scalpel blade if fluid is not oozing freely from lesion.
2. Tear open the top of the sterile swab transport unit, being careful to touch only the cap of the swab. Remove and discard the cap of the enclosed transport unit.
3. Remove the sterile swab, handling only the cap of the swab.
4. Holding the swab firmly, take the culture by touching the sterile fiber end to the area to be cultured. If it is a deep lesion, push the swab gently into the deepest portion. Remove the swab from the culture site. Do not set the swab down.
5. Insert swab, fiber end first, deep into the medium so that the fiber end is fully immersed. Cap securely.
6. Apply fresh dressings if needed.
7. Label the swab transport unit with patient's identification sticker from Test Request.
8. Fill out the Test Request following the Microbiology Instruction Sheet. Keep at room temperature. Ship warm with RML courier.
   NOTE: If culturette is used, use the same procedure but remember to crush ampule after the specimen is collected and swab is returned to holder. SWAB MUST NOT BE ALLOWED TO REMAIN DRY.

ANAEROBIC CULTURE COLLECTION PROCEDURE

EQUIPMENT:

1. If specimen is to be aspirated:
   • A.C.T. I tube at room temperature.
   • Sterile 12 cc syringe with 22 gauge needle.
   • Extra sterile needle.
   • Iodophor swab.
   • Alcohol pad.
   • Sterile disposable gloves, if desired.
2. If unable to aspirate specimen:
   • A.C.T. I tube at room temperature.
   • Sterile cotton swabs.
   • Sterile saline.
   • Sterile gauze sponges.
   • Sterile scalpel blade.
   • Sterile disposable gloves, if desired.

SPECIMENS FOR WHICH ANAEROBIC CULTURE IS RECOMMENDED:

• Body fluids — Blood, ascitic fluid, synovial fluid, thoracentesis, pleural fluid, transudates, cerebrospinal fluid (CSF)
• Exudates — Aspirated pus from deep wounds or abscesses
• Surgical specimens — Gallbladder, etc.
• Respiratory — Transtracheal aspirate only
• Genital specimens — Placenta, Bartholin's gland, culdocentesis, endometrial, fallopian tube, septic abortion, prostatic or seminal fluids

SPECIMENS FOR WHICH ANAEROBIC CULTURE IS NOT DONE OR RECOMMENDED:

• Urines, voided or catheterized
• Stools
• Expectorated sputum or bronchial washings; throat; tonsil; nose; ear
• Vaginal, cervical and urethral specimens

PROCEDURE:

1. Explain procedure to patient to reduce anxiety.
2. If site is not an open wound, prepare the surrounding skin by wiping with Iodophor swab, then alcohol swab. Allow to dry. This reduces contamination of specimen with skin flora.
3. If source is an abscess with a large amount of pus or is a fluid:
   • Wipe rubber top of A.C.T. I tube with alcohol.
   • Aspirate pus or fluid to be cultured with sterile syringe and needle.
   • Expel any air present in the syringe but do not spray the environment.
   • Inject specimen into the A.C.T. I tube.
   • Label transport medium with patient's identification sticker from Test Request.
   • Fill out Test Request form following the Microbiology Instruction Sheet and send specimen to the laboratory. Keep at room temperature.
4. If source is an open abscess or wound or has little pus:
   • Remove dressings, if present. Wash area gently with sterile saline and sterile cotton gauze to remove encrusted material, if present.
   • Open lesion carefully with sterile scalpel blade if fluid is not oozing freely from lesion.
   • Loosen the screw-top of the A.C.T. I tube.
   • Tear open the top of the sterile cotton swab package and remove one of the sterile cotton tip swabs, handling only the stick end.
   • Holding the swab firmly, obtain the culture by pushing the swab to the deepest portion of the wound. Remove the swab from the culture site. Do not set the swab down.
   • With the little finger of the right hand, carefully remove top of transport medium and insert the swab, cotton end first, deep into the medium so that the cotton end is fully immersed.
   • Break off the stick of the swab just below the level of the neck of the transport vial and replace the screw cap of the transport vial. Replace dressings if they were present with fresh dressings.
   • Label the transport vial with patient's identification sticker from Test Request.
   • Fill out the Test Request form following the Microbiology Instruction Sheet. Keep at room temperature and ship with RML courier.

EQUIPMENT:

• Sterile 20 cc syringe with 20-22 gauge needle
• One Chloroprep Sepp ampule
• Three alcohol pads
• Tourniquet
• For adults, one aerobic BacT/Alert bottle and one anaerobic BacT/Alert bottle
• For children (under 13 years), one aerobic BacT/Alert bottle

PROCEDURE:

1. Explain procedure to patient to alleviate anxiety.
2. Apply tourniquet and locate vein. Remove tourniquet to prepare site and replace before venipuncture.
3. Start in the center of the area where the venipuncture will be made and scrub with moderate pressure with the Chloroprepp ampule, working outward in a circular motion.
4. ALLOW THE AREA TO DRY.
5. DO NOT TOUCH THE VENIPUNCTURE SITE ONCE IT HAS BEEN PREPARED UNLESS YOUR FINGER HAS BEEN SIMILARLY PREPARED. Think of this as preparation of a surgical site. Contamination of blood cultures from skin flora have significant impact on patients resulting in unnecessary antibiotic therapy and unnecessary hospitalization time.
6. Wipe the rubber top of the blood culture bottle(s) with the Choroprepp.
7. Replace tourniquet and perform venipuncture, obtaining at least 20 cc of blood*. Efficiency of blood culture procedure in demonstrating bacteria in the blood is dependent upon the volume of blood cultured.
   *NOTE: In children 2-13 years of age, 10 ml should be drawn, in those 2 months to 2 years, 3-5 ml and in those under 2 months, 1-3 ml.
8. Inject 10 cc of blood into each BacT/Alert bottle. DO NOT INTRODUCE AIR INTO THE BOTTLES. In children 2-13 years of age, inject only aerobic BacT/Alert bottle (10 cc). In those under 2 years of age, inject entire sample into aerobic BacT/Alert bottle. If limited blood (less than 5 ml) is obtained on any patient, inject only in aerobic BacT/Alert bottle.*
   *NOTE: Recent studies show that it is NOT necessary to replace the needle used for the venipuncture by a fresh needle before injecting the blood into the bottles. AS LONG AS THE SKIN PREPARATION IS ADEQUATE, there should not be significant carryover of bacteria from the patient's skin. If the first attempt to obtain blood is successful, a new sterile needle must be used for the second venipuncture.
9. Label all bottles with patient identification.
10. Fill out the RML Test Request following the Microbiology Instruction Sheet.
11. Hold bottles at room temperature or incubate at 35 degrees C if possible. Transport to laboratory without delay. NOTE: If additional laboratory work is required, inject blood culture bottles first before entering any other tube.

ENDOCERVICAL SWAB:

1. Remove excess mucus from cervical os and surrounding mucosa using cleaning swab (white shaft swab in package with red printing). DISCARD THIS SWAB.
2. Insert specimen collection swab (blue shaft swab in package with green printing) into endocervical canal.
3. Gently rotate swab clockwise for 10-30 seconds in endocervical canal to ensure adequate sampling.
4. Withdraw swab carefully; avoid any contact with vaginal mucosa.
5. Remove cap from swab specimen transport tube and immediately place specimen collection swab into specimen transport tube.
6. Carefully break swab shaft on scoreline; use care to avoid splashing contents.
7. Re-cap swab specimen transport tube tightly and label with patient identification.
8. Store at 2-30 degrees C (refrigerated or room temperature).

MALE URETHRAL SWAB:

NOTE: Patient should not have urinated for at least one hour prior to specimen collection.

1. Insert specimen collection swab (blue shaft in package with green printing) 2-4 cm into urethra.
2. Gently rotate swab clockwise for 2-3 seconds in urethra to ensure adequate sampling.
3. Withdraw swab carefully.
4. Remove cap from swab specimen transport tube and immediately place specimen collection swab into specimen transport tube.
5. Carefully break swab shaft at scoreline; use care to avoid splashing contents.
6. Re-cap swab specimen transport tube tightly and label with patient identification.
7. Store at 2-30 degrees C (refrigerated or room temperature).

URINE - MALE AND FEMALE:

NOTE: Patient should not have urinated for at least one hour prior to specimen collection.

1. Direct patient to provide first-catch urine (approximately 20-30 ml of initial urine stream) into urine collection cup free of any preservatives. Collection of larger volumes of urine may result in specimen dilution that may reduce test sensitivity. Female patients should not cleanse labial area prior to providing specimen.
2. Remove cap from urine specimen transport tube and transfer 2 ml of urine into urine specimen transport tube using disposable pipette provided. The correct volume of urine has been added when fluid level is between black fill lines on urine specimen transport tube label.
3. Re-cap urine specimen transport tube tightly and label with patient identification.
4. Urine samples that are collected without the transport tube may be submitted in a clean urine container if it arrives in the laboratory within 24 hours of collection.

SPECIMEN TRANSPORT AND STORAGE:

1. After collection, transport the processed urine specimens in the Gen-Probe APTIMA Combo 2 Assay urine specimen transport tube at 2-30 degrees C until tested. Processed urine specimens should be assayed with the APTIMA Combo 2 Assay within 30 days of collection. If longer storage is needed, freeze at -20 degrees C to -70 degrees C for up to 90 days after collection.
2. Urine samples that are still in primary collection container must be transported to laboratory at 2-30 degrees C. Transfer urine samples into APTIMA Combo 2 Assay urine specimen transport tube within 24 hours of collection. Store at 2-30 degrees C and test within 30 days of collection.

The urethral/conjunctival swab should be inserted 2-4 cm into the urethra using a rotating motion. Once inserted, rotate the swab gently for 2-3 seconds using sufficient pressure to ensure contact with all urethral surfaces. Withdraw swab, place in transport, break swab at score line, cap securely and transport as above.

NOTES:

• Specimens submitted for probe must be collected using GenProbe swabs and transport. Use of other swab will result in rejection of specimens.
• Once sample is placed in transport, it is non-viable for culture. Results are limited to detection of GC and/or chlamydia nucleic acid.
• Specimens from cases of criminal sexual conduct must be tested by culture techniques only, according to CDC.
• Specimens from sites other than urethra, cervix and conjunctiva have not been evaluated for their performance by this method; results may not be reliable. Culture methods should be used if other sites need to be tested.
• A single sample can be tested for both GC and chlamydia; either test may be requested separately.

SPECIMEN REQUIRED — 2-3 ml minimum stool

EQUIPMENT NEEDED — One sterile urine container or clean, dry plastic margarine tub

PROCEDURE:

1. Collect stool sample in container. Avoid contamination with urine. Do not contaminate with toilet paper. CAP SECURELY.
2. Label with patient's identification sticker from Test Request.
3. Fill out Test Request following the Microbiology Instruction Sheet.
4. Refrigerate specimen at 2-8 degrees C.
5. Ship on ICE with RML courier.

NOTE: Testing of two separate stool passages is recommended for maximum sensitivity in detection.

SPECIMEN REQUIRED — Hair, skin scrapings, nail trimmings, swab or air dried smear of vaginal discharge.

MATERIALS REQUIRED:

• Sterile scalpel blade for skin scrapings and DermaPak for transport
• Sterile forceps to grasp hair and DermaPak for transport
• UV (Woods) lamp to visualize infected hairs
• Sterile scissors to trim infected nails and sterile container for transport
• Sterile swab/transport unit for vaginal discharge or slide and plain swab for collection
• Disposable gloves

PROCEDURE:

1. Wear disposable gloves.
2. Explain procedure to patient to eliminate anxiety.
3. If a skin lesion, carefully unwrap scalpel blade and gently scrape the advancing margin of the lesion with the blade. Do not scrape so hard as to draw blood. Collect specimen by holding DermaPak open under lesion as scraping is performed. Remove scraping left on blade by rubbing off on inside of DermaPak. Close DermaPak, label with numbered sticker from Test Request and place in plastic bag which held DermaPak.
4. If hair is infected, use UV light to help visualize infected hair; pluck those which fluoresce, using sterile forceps and place on open DermaPak. (NOTE: Not all fungal infections will result in fluorescence.) Close DermaPak, label with numbered sticker and place in plastic bag which held DermaPak.
5. If nails are infected, carefully trim a portion of the nail into a sterile container. Label with numbered sticker from Test Request.
6. If vaginal discharge is to be examined for fungal elements, submit a swab of the discharge using the routine swab/transport unit. Alternately, an air dried smear of vaginal discharge can be submitted in a cardboard holder. Label either with numbered sticker from Test Request.
7. Complete patient information on Test Request and order Test #979, Direct Fungal Prep. Hold the sample at room temperature until transport to the laboratory for testing.

NOTE: Processing techniques appropriate to the sample will be performed. Thick specimens such as nails will be dissolved using KOH; swabs from vaginal discharge will be stained with a fluorescent dye which is very sensitive and specific for fungal cell walls. This stain cannot be used on samples in KOH because of pH alteration. If observed, clue cells will be reported from vaginal secretion.

SPECIMEN REQUIRED — Hair, skin scrapings or nail trimmings.

EQUIPMENT NEEDED:

• Sterile scalpel blade, fine
• Forceps or sterile scissors
• Sterile petri dish or fresh sheet of typing paper
• Disposable gloves, if needed
• STERILE, EMPTY tube with plug or cap for transporting specimen

PROCEDURE:

NOTE: Plain sterile red top vacutainer tube is acceptable but NOT an SST (barrier) tube for transporting specimen.

1. Explain procedure to patient to alleviate anxiety.
2. Unwrap scalpel blade being careful not to touch blade portion.
   • Skin lesion — Gently scrape skin at advancing margin of lesion with blade. Do not scrape hard enough to draw blood. Collect scrapings by holding petri dish or paper under lesion as scraping is performed. Transfer carefully to empty tube.
   • Hair — Gently pluck several infected hairs with sterile forceps and transfer carefully to empty tube.
   • Nails — Carefully trim portion of infected nail with sterile scissors, collecting trimmings in sterile petri dish or on fresh paper. Transfer carefully to sterile empty tube.
3. Cap tube securely. Keep at room temperature.
4. Label tube with patient's identification sticker from Test Request.
5. Fill out Test Request following the Microbiology Instruction Sheet.
6. Ship to RML with courier.

Sputum, CSF, tissues, etc. — Collect and transport as specified on MICRO section.

GC (Neisseria gonorrhoeae) only

EQUIPMENT NEEDED — Collect the specimen with a swab transport unit (or culturette).

PROCEDURE, FEMALE:

1. Cervix (the best site to culture) — Insert the speculum, remove cervical mucus with cotton balls or swab and discard. Insert sterile Dacron swab from transport unit (or culturette swab) into endocervical canal. Move from side-to-side gently. Recap and label. Hold at room temperature and transport to laboratory.
2. Anal culture (most likely place to be positive if cervix is negative) — Insert sterile Dacron swab from transport unit (or culturette swab) one inch into anal canal (if swab is inadvertently pushed into feces, discard and use fresh swab). Move swab from side-to-side in anal canal to sample crypts. Recap and label. Hold at room temperature and transport to laboratory.
3. Vaginal or urethral culture (post-hysterectomy or in children).
   • Urethral — Strip urethra towards the orifice to express exudate. Collect on swab. Recap swab and label. Hold at room temperature and transport to laboratory.
   • Vaginal — Use speculum to obtain specimen from posterior vaginal vault or from vaginal orifice if hymen is intact. Collect specimen with swab. Recap and label. Hold at room temperature and transport to laboratory.

PROCEDURE, MALE:

1. Urethra — Use sterile N/P swab transport unit to obtain specimen from anterior urethra by gently scraping mucosa or milking exudate. Recap and label. Hold at room temperature and transport to laboratory.
2. Anal — Obtain as for female.

ROUTINE GENITAL CULTURE

EQUIPMENT NEEDED — Swab transport unit.

Obtain specimen as described under "GC Culture".

Bacterial pathogens normally sought in routine culture include Neisseria gonorrhoeae (GC), yeast, Strep. agalactiae (Group B), Staph. aureus, Strep. pneumoniae and Haemophilus influenzae.

As routine culture includes yeast and GC, those cultures need not be ordered separately if routine is ordered. NOT FOR THE DIAGNOSIS OF HERPES OR CHLAMYDIAE (see those protocols).

Anaerobic culture generally is inappropriate for specimens from routine genital sources due to the presence of normal anaerobic flora.

Smear performed as part of routine culture will indicate if clue cells (suggestive of bacterial vaginosis) are present.

GENITAL GRP B SCREEN

EQUIPMENT NEEDED — Swab transport unit

Collect genital or rectal specimen as described under "GC Culture".

Grp B strep. (Strep. agalactiae) only will be sought. As routine culture includes Grp B strep. culture, this need not be ordered separately when a routine genital culture is ordered.

HERPES VIRUS CULTURE COLLECTION PROCEDURE

EQUIPMENT NEEDED:

• One viral transport unit (supplied by RML), refrigerated
• One sterile scalpel blade
• One pair disposable gloves

COLLECTION FROM VESICULAR LESION:

NOTE: Collection and handling of specimens is of the utmost importance in successful virus isolation. Specimens should be collected as early as possible in the course of the disease, preferably from a fresh, NOT a crusted lesion.

PROCEDURE:

1. Rupture early-stage lesions with a sterile scalpel blade.
2. Gently swab the opened vesicle to collect fluid and cells from the base of the lesion using the sterile Dacron swab provided, NOT CALCIUM ALGINATE OR WOODEN STICK SWABS as these may be toxic to viruses.
3. Place the swab in the viral transport medium provided. Carefully break off the swab avoiding the generation of aerosols which may be infectious. Replace cap SECURELY.
4. Label tube with patient's identification sticker from Test Request. Please indicate on the Test Request under "Remarks" the date of onset and any local or systemic symptoms, as well as the source of the specimen.
5. Hold and transport specimen at refrigerator temperature. Specimens may be refrigerated overnight but delay in processing reduces the chance of recovery of viruses. Ship on ICE with RML courier.

COLLECTION OF SPECIMEN FOR PRENATAL SCREENING:

NOTE: When no lesions are obvious in the pregnant female with a history of genital lesions, the American Academy of OB/GYN recommends three blind cervical cultures be collected during the last 6-8 weeks of pregnancy to detect asymptomatic shedding. Demonstration of Herpes simplex in culture from the cervix of a pregnant female in the last trimester may be an indication of the need for a Cesarean section.

PROCEDURE:

1. Using the Dacron swab provided, collect the specimen from the endocervical canal.
2. Place the swab in the viral transport medium provided. Carefully break off the swab avoiding the generation of aerosols which may be infectious. Replace cap securely.
3. Label the tube with patient's identification sticker from Test Request. Please indicate on the Test Request under "Remarks" the date of onset and any local symptoms, if applicable, as well as the source of the specimen.
4. Hold and transport specimen at refrigerator temperature. Specimen may be refrigerated overnight but delay in processing reduces the chance of recovery of viruses. Ship on ICE with RML courier.

NOTE: Please keep transport medium refrigerated in storage and after use.

Should any questions arise as to collection or interpretation of results, please contact our Microbiology Department Supervisor.

SPECIMEN REQUIRED: Secretions of the nasopharynx are superior for the detection of Influenza A or B and can be submitted on a nasopharyngeal swab or as an aspirate.

NASOPHARYNGEAL SWAB

MATERIALS REQUIRED:

• Dacron or Rayon nasopharyngeal swab (DO NOT use calcium