M i c r o b i o l o g y  C o l l e c t i o n  &  H a n d l i n g   G u i d e l i n e s

General Information
Quality microbiology results depend on both the client and the laboratory. Successful isolation of potential pathogens is a factor of specimen selection and collection, proper transport and timely delivery as well as excellent microbiology practices within the testing laboratory. It is critical to refer to specimen submission requirements for specific cultures in the Test Directory to assure that appropriate transport media is used. Please contact Client Services for detailed information.

Test Request Form: Submit a completed Test Request Form (PDF) for each culture requested. Information regarding the patient, the specimen, collection time and date, clinical history, symptoms and diagnosis, antimicrobial therapy (or use of any other chemotherapeutic agents) and any suspected organism(s) is essential for the optimal and appropriate processing of the specimen. If an organism is sent for identification, the suspected organism or type must also be indicated. Clearly indicate the source and type of culture requested.

Specimen Containers: Shipping containers, sterile specimen containers, transport media and swabs are available through Client Services. Specimens are acceptable for processing only when collected and submitted in the appropriate container. Specimen containers must be tightened securely to obviate any leakage.

Labeling: Clearly print the patient's complete name (as listed on the test request form) on the culturette or other specimen transport media. (Labeling of only the outside container is not acceptable).

Transport: Deliver specimens to the laboratory within 24 hours. Prompt processing minimizes loss in viability of potential pathogens and insures a more accurate appraisal of flora present.

Results Reporting: Preliminary and final results for Microbiology are included on the patient report. Preliminary results will be called to the client on any positive, significant culture (e.g., blood, CSF, sterile body fluid and Salmonella or Shigella isolates) or stain. Other results will be called to the client if the client writes "Call Results" on the test request form and indicates a phone number to which results should be called. Results or interpretations of results may be obtained by contacting Client Services.

Rejection Criteria: Unacceptable specimens may result in test cancellation or delay. The Client Services staff will contact the client with such information and will also document the fact on the report. Listed below are general causes of such action. 

1. Incomplete patient information on the test request form 

2. Missing source 

3. Improper or missing labeling on specimen 

4. Conflicting labeling information between the specimen and the test request form 

5. Inappropriate specimen for culture requested 

6. Improper specimen container or transport media 

7. Leaking or spilled specimens 

8. Specimen age 

Clinical Virology
An adequate specimen, properly collected from an appropriate site is imperative to obtain significant and timely results. Any delays in transport or improper storage prior to transport will also adversely affect the potential for virus recovery. The requesting client must indicate the specific etiologic agent(s) suspected. The client will be notified, by telephone, of any rejected specimens or any specimens contaminated or toxic to tissue culture. Repeat collection of the specimen will be requested.

Collection & Transportation of Specimens for Virus Isolation:

• Collection Materials 

  • M4 Multi-Microbe Transport System contains a tube with 3mL viral transport media and 2 collection swabs. The small swab on a mesh handle should be used for nasopharyngeal or urethral specimen collection. The M4 Transport System kits are available upon request from Client Services. Bacterial Culturette swabs may be used as an alternative collection system when the optimal transport system is not available. M4 Transport Media must be stored at refrigerated temperatures. Observe the expiration dates on all media. Specimens should be stored and transported at refrigerated temperatures.

  • HPV (Digene) Collection Kits contain collection swabs and tubes specifically for the detection of HPV DNA. The HPV collection kits are available upon request from Client Services. The HPV collection kits may be stored at room temperature. Specimens should be stored and transported at refrigerated temperatures. Cervical swabs may also be collected in Cytyc ThinPrep™, PreservCyt™ solution. Transport solution at room temperature or refrigerated.

  • Blood for viral isolation should be collected in a 10mL (lavender top) vacutainer tube. Transport at room temperature.

  • Sterile Containers should be used for Stool, Urine, and other Body Fluids. 

  These specimens must NOT be diluted with M4 Transport Media. Specimens should be stored and transported at refrigerated temperatures.
  

• Transport Conditions:

  • DO NOT FREEZE any specimens being submitted for Virus Isolation. The only exceptions are stool specimens for Rotavirus Detection; these specimens may be transported frozen or at refrigerated temperatures.

  • Refrigerate all specimens after collection and during transport EXCEPT BLOOD. Blood specimens should be kept at room temperatures.

  • Deliver all specimens to the laboratory within 24 hours of collection for the optimal recover of viruses.

Collection Instructions by Specimen Type:

Conditions for Rejection of Specimens: Specimens which may be rejected will be held until the ordering physician or laboratory can be contacted to determine if a repeat specimen can be obtained. The Virology Laboratory will reject specimens for the following reasons:

Specimen Guide for Specific Viruses:

Viral Culture Panels: The following panels are available for the identification of various viral infections. Please see the Test Directory for specimen requirements.

*CSF requests for HSV, VZV and CMV should be ordered as PCR. Cultures are not performed.

Retrovirology
Tests for the detection of serum antibodies to Human Immunodeficiency Virus (HIV) are available at the Reference Laboratory at the Cleveland Clinic. Antibodies are detected utilizing an enzyme-like immunosorbent assay (ELISA). Supplementary testing by Western Blot Analysis is also available and will be done automatically for all sera that are reactive by ELISA. An additional fee will be charged for the Western Blot. Both tests detect antibodies to HIV and are approved by the FDA for diagnosis of HIV infection; they are not tests for the disease entity known as Acquired Immunodeficiency Syndrome (AIDS) or related conditions. Information regarding HIV serologic status of individuals who engage in behavior that places them at risk for acquiring HIV infection is of benefit to a physician in his/her medical assessment of such individuals. A test result of reactive does not mean that an individual being tested has AIDS or will develop it; it does mean that in all likelihood, the individual has been exposed to HIV. As with other serologic tests, careful interpretation is required. False positive reactions can occur; conversely, a test result of non-reactive does not categorically eliminate the possibility that infection has occurred. Individuals who are carriers of HIV but are serologically non-reactive for antibodies to the virus have been reported. Moreover, serum sampled after infection but prior to seroconversion would also test non-reactive. For these and other reasons, careful pre-test and post-test counseling and follow-up of individuals who are tested must be done by the physician, and extreme care must be taken to ensure confidentiality.

Parasitology
The Reference Laboratory at the Cleveland Clinic offers diagnostic procedures for blood, tissue and enteric parasitic diseases, occult blood testing, Chlamydia, Mycoplasma and qualitative testing on stool. Recovery of parasites is critically dependent upon the quality of the specimen received by the laboratory. Refer to specific collection instructions for each test requested (Test Directory).

Criteria for Collection of Stool Specimens for Ova and Parasites: 

• Unless otherwise specifically stated by the physician, specimens should be 24 hours apart. If the circumstances require it, a 12-hour minimum will be acceptable. 

• Ova and parasite exams are not recommended on patients who have been hospitalized for over three (3) days. Almost no parasites are recovered from these patients. Some exceptions to this rule are: patients with diarrhea on admission who were never cultured or immunosuppressed patients with intermittent shedding in whom Giardia, amebiasis or Cryptosporidium is suspected. 

• Stool specimens may be taken after proctoscopic exams. Procto specimens are not equivalent to normal bowel movements. These types of specimens are processed by different methods; therefore, DO NOT equate one with the other. 

• Specimens will be rejected for Ova and Parasite determination if taken after a barium enema, after the administration of castor oil, (i.e., for lower colon x-rays or urograms, etc.) or after the administration of other laxatives or suppositories. Saline enema specimens are acceptable for Ova and Parasites, but must be noted as "Saline enema specimen." 

• Ova and Parasite specimens must be properly collected and placed in the O & P kits available through Client Services. Containers or specimens lined with plastic, tissue paper, newspaper, etc. are unacceptable and will be rejected. 

• Do not refrigerate or freeze specimens. 

• To rule out parasitic infection, screening one fresh specimen daily for three days is advisable. 

• Consult the Test Directory for the specific procedure desired for more detailed information. 

Antibiotic Susceptibility Testing
Antibiotic susceptibility testing will be performed routinely using the broth microdilution method that gives results in terms of minimum inhibitory concentration (MIC). 

The MIC is defined as the lowest concentration of an antibiotic which will inhibit the in vitro growth of an infectious organism. The results of microdilution susceptibility tests are reported in micrograms or units per mL. The interpretation of these in vitro data is based on the achievable concentration of the antibiotic in vivo, which may vary depending on dose, routine of administration, degree of protein binding, site of infection, age and weight of the patient, state of health of the patient, and other factors.

Reporting of MICs can provide the physician with precise information regarding the degree of susceptibility of the infecting organism. When this information is coupled with the physician's knowledge of the site and severity of the infection, as well as the pharmacology of antibiotics, a rational choice of the most appropriate antibiotic can be made to suit the individual patient. With the quantitative MIC: 

1. susceptibility can be determined for dosages and routes of administration other than those usually prescribed, and 

2. susceptibility can more accurately be related to the achievable antibiotic concentration in urine, bile, CSF, and other body fluids which may vary widely from the achievable concentration in serum.

As a general rule, for an antibiotic to be considered for use, the in vivo concentration achievable at the site of infection should be a minimum of 2-4 times the MIC. Although the MIC provides useful data in the selection of an antibiotic, the clinician must also consider antibiotic pharmacology, mode of action, volume of distribution, toxicity, allergicity, clinical efficacy, and infection pathology.

Antibiotic Assays: Assays for the following individual antibiotic and antibiotic combinations will be performed:

Any combinations not listed are considered experimental and will be tested on a time-available basis. The laboratory must be aware of all antibiotics given to the patient within the week preceding the time blood is drawn for assay so that appropriate steps can be taken to eliminate or nullify the effects of interfering drugs.

Antibiotic Assays Monitoring: Assays for the following individual antibiotic and antibiotic combinations will be performed and may be tested in the presence of any antibiotic: Amikacin (Amik); Gentamicin (Genta); Tobramycin (Tobra); and Vancomycin (Vanco)

Factors Affecting Monitoring of Selected Antibiotics:

P = Peak Level;    T = Trough Level

These recommendations are valid when dosage interval is no greater than twice the usual dosage interval. Blood for peak levels should be drawn 30 minutes after completion of an IV infusion, 1 hour after an IM dose, and 1 to 2 hours after an oral dose. (Adapted from Hermans, P.; Anhalt, J.P.; Washington, J.A.-I Pocket Manual of Antimicrobial Agents, B.C. Decker Inc., Philadelphia 1987. )

Culture Types and Guidelines
Complete a Test Request Form, indicating the specific source of the specimen, diagnosis and organism(s) suspected. Label the specimen appropriately. Record the date and time the specimen was collected.

Blood Cultures: The detection of microorganisms in blood has great diagnostic and prognostic importance. A completed test request form (particularly time of collection, diagnosis, and antimicrobial therapy) is essential for timely and appropriate laboratory processing of the specimen.

All routine blood cultures are screened for aerobic and anaerobic bacterial and fungal isolates. All organisms isolated from blood cultures will be identified, susceptibility tests performed, if appropriate, and stock cultures made, unless the isolate is considered a skin contaminant. Stock cultures will be held for 30 days. Organisms which may be contaminants are coagulase negative Staphylococcus species, Bacillus species, diphtheroid bacilli and viridans Streptococcus species.

Blood Cultures from patients suspected of having Brucella must be specifically requested as special cultures. (See the culture listings for the specific organisms for further details).

The following guidelines are provided in order to aid in determination of how many cultures to draw, when they should be drawn, and how they should be obtained:

1. In most adult infections, cultures of two to three separate venipunctures (20-30mL per venipuncture), initially, are sufficient. Studies have shown the following positive blood culture yields within a 24-hour period in patients without endocarditis:

   First Venipuncture (Set):	80%
   First and Second Venipunctures (Set):	90%
   First, Second and Third Venipunctures (Set):	99%

   Based on these results, the laboratory will not accept more than four blood culture sets per 24 hours per patient. 

2. In suspected acute sepsis, meningitis, osteomyelitis, arthritis, or acute untreated bacterial pneumonia, obtain two blood culture sets (from two separate venipuncture sites) before starting therapy. 

3. For suspected infective endocarditis. 

   1. Acute: Obtain three blood culture sets with three separate venipunctures during the first one to two hours of evaluation. 

   2. Subacute: Obtain three blood culture sets on Day 1 (15 minutes or more apart). If all three are negative at 24 hours, obtain three more. 

4. There exists a direct correlation between the volume of blood cultured and the recovery rate of clinically significant organisms. For adult patients, each blood culture bottle should receive 10mL of blood. Isolators should receive 10mL as indicated on the tube. For pediatric patients, one to five mL of blood injected into one aerobic blood culture bottle is sufficient. 

5. Specimen Collection: After placing a tourniquet on the patient's arm, select a suitable vein. Once the vein has been found, cleanse the area with the series of 2% iodine, 70% alcohol, iodine and alcohol. Without touching the site of the venipuncture with the fingers, draw 20mL of blood into a sterile syringe. Inject 10mL of the specimen into each of two culture bottles after cleansing the stoppers with 2% iodine. Invert the containers several times to insure thorough mixture of the contents. (Blood culture systems are available through Client Services). For pediatric patients, write the volume of blood inoculated on the label. 

6. Blood Cultures from patients suspected of having Histoplasma capsulatum, Brucella, Bartonella, Legionella or Mycobacterium must be specifically requested as special cultures and must be collected in a lysis-centrifugation (Isolator™) tube. (See the culture listings for the specific organisms for further details.) 

Urine Cultures: Submit specimens in urine culture transport tubes provided by the Reference Laboratory at the Cleveland Clinic. Indicate whether the specimen is from a clean catch or a catheter.

Patient Collection Guidelines: Clean Catch

Patient Collection Guidelines: Indwelling Catheter 
Obtain the specimen with a needle and syringe. Select the puncture site 1-2 inches away from the catheter tube entry point. Cleanse the area to be punctured with 70% alcohol. Aspirate exactly 5mL of urine with a sterile needle and syringe. Disinfect the rubber stopper and aseptically transfer the specimen to the urine transport tube provided. Specimens obtained from the collection bag are not suitable for analysis. Foley tips will not be accepted.

Urine Culture Transport: Prevention of contamination by normal vaginal, perineal, and anterior urethral flora is the most important consideration for collection of a clinically relevant urine specimen. (See the Patient Collection Guidelines above for collection of a urine specimen). Unpreserved urine is an excellent growth medium for most bacteria. Unless urine is preserved during transport, bacteria may multiply, causing colony counts to be erroneously high. The maintenance medium in the transport kit prevents rapid multiplication of bacteria in the urine during shipment. 

Instructions for use of Urine Culture Transport System:

1. Obtain the urine specimen from the patient in a clean cup. 

2. Open the pouch and remove the transfer device and tube. 

3. Submerge the pipette of the transfer device to the bottom of the urine container. The container may be tipped at an angle if the volume of the urine is limited. 

4. Place the transport tube in the holder portion of the transfer device and push it down as far as it will go, puncturing the stopper. 

5. Hold the tube and transfer device in position until the urine stops flowing into the tube. 

6. Remove the transport tube from the transfer device and shake the tube vigorously. 

7. Discard the transfer device and remaining cup of urine into appropriate biohazard disposal containers. 

Precaution: The transport tube must be filled to the minimum line indicated on the tube. A tube that is not filled at least to this line is unacceptable for culture.

Respiratory Culture: The following collection sites are considered respiratory specimens: mouth, nose, nasopharyngeal swabs, throat sputum, bronchial and tracheal aspirations, bronchoalveolar lavage (BAL) and Bartlett Fiberoptic Wimberly (BFW) brush. All specimens from these sites will be screened for fast-growing aerobic pathogenic organisms and for Beta Strep Group A.

The following organisms are examples of those not isolated by routine culture: Neisseria gonorrhoeae, Corynebacterium diphtheriae and Bordetella species. Specimens from the nose are the only respiratory specimens routinely screened for Haemophilus species. For information on culture of non-routine organisms, see the culture listing for the specific organism.

Stool Cultures: Refer to the Test Directory for collection information. Specimens must be submitted in transport media provided by the laboratory.

Routine Bacterial Cultures: Complete a Test Request Form (PDF), indicating the specific source of the specimen, diagnosis and organism(s) suspected. Label the specimen appropriately. Record the date and time the specimen was collected.

Miscellaneous Cultures: 

• Specimens from normally sterile sites (such as body fluids, surgical specimens, joint fluids, etc.) will be cultured for aerobic pathogenic organisms. Anaerobic pathogens may be detected by routine cultures. However, be aware of the special requirements for the isolation of anaerobes. If an anaerobic organism is suspected, a specimen should be submitted for anaerobic culture. 

• Bone marrow, eye swabs, joint fluids and cerebrospinal fluids are routinely screened for fast-growing aerobic pathogenic bacteria including Haemophilus species and pathogenic Neisseria species. 

• Body fluids (exclusive of joint or cerebrospinal fluids) and surgical specimens will be routinely screened for fast-growing aerobic pathogens. 

• Genital tract specimens are tested for Neisseria gonorrhoeae and/or Chlamydia trachomatis by amplification. If Haemophilus ducreyi is suspected, a special request must be made. In cases of suspected toxic shock syndrome, the tentative diagnosis should be placed on the requisition form so that isolates of Staphylococcus aureus may be saved for TSST-1 testing. Vaginal specimens must be accompanied by a request for examination for a specific organism (i.e. group B streptococci, Candida, Trichomonias vaginalis) or for gram stain, which may be examined for the presence of clue cells, curved bacteria, relative proportion of gram-positive bacilli resembling lactobacilli vs. Gram-negative bacilli. Culture for Gardnerella vaginalis is neither sensitive nor specific for the diagnosis of bacterial vaginosis and will not be done. 

• Specimens from wounds, ulcers, and drainages will be screened for nonfastidious, fast-growing aerobic organisms. If an anaerobe is suspected, a specimen must be properly submitted and an anaerobic culture specifically ordered. 

Nose or Nasopharyngeal Swabs: Collect in a manner to avoid contamination by adjacent structures.

Throat Specimens: Collect in a manner to avoid contamination by mouth and tongue. Throat specimens will be screened for Group A Strep only. Throat specimens for Neisseria ganorrohoeae, Bordetella pertussis or Corynebacterium diphtheriae will be screened only for that pathogen when specifically requested. Collect in a manner to avoid contamination by mouth and tongue.

Sputum Specimens: Collect by instructing the patient to remove dentures, rinse mouth, gargle with water and cough deeply, expectorating into appropriate collection container. All specimens labeled "sputum" that are consistent on cytologic examination with saliva or upper respiratory secretions will be rejected. Rejection or acceptance will be based on microscopic examination for the absence of/or minimal numbers of squamous epithelial cells.

Tracheal and Bronchial Aspirations: Collect by physician to avoid contamination with adjacent structures.

Fungus: Provide complete information on the Test Request Form (PDF) to insure that the specimen is cultured for all organisms (fungi/yeast) suspected. Additional patient history may be helpful. Include the patient occupation, history of travel or residence abroad, and any animal contacts. A single specimen may be cultured for both bacteria and fungi. Generally, a direct fungal smear exam will be performed on all specimens submitted for fungal cultures. 

Yeast: Requirements are the same as for a fungal culture. Urine, vaginal, throat and stool requests are routinely processed for a yeast culture, even if ordered as a fungal culture.

Anaerobes: Cultures for fastidious anaerobes will not be performed on urine (nonsuprapubic aspirate) or specimens from body sites that have anaerobes as part of the normal flora (stool, skin, vagina/cervix, mouth, throat, septum, etc.). Any material collected from areas with no normal flora (body fluids, deep abscess exudate, tissue biopsies, transtracheal aspirates, etc.) is acceptable for anaerobic culture.

Specimens for anaerobic culture must be collected appropriately and transported in an anaerobic environment (anaerobe vial). Aspirated specimens, in general, are preferred. Swabs are unacceptable for anaerobe culture. Contact Client Services prior to collection of the specimen for consultation on collection procedures, containers, or transport.

Acid-Fast Bacillus: All cultures and stains for AFB are performed at The Cleveland Clinic. Acid-fast stain results are generally available 24-48 hours from receipt of sample. Positive stains and/or culture results are called to the client. Transport specimens in sterile, leak-proof containers and place in appropriate shipping containers. Dry specimens or spilled-leaking specimens are not acceptable for AFB culture. Specimens submitted on swabs or sputums of low volume are discouraged for AFB culture because the yield is significantly decreased. Gastric contents must be neutralized with sodium carbonate or another alkaline buffer salt before transport. 

AFB Turnaround Times (General Guide)

Respiratory specimens may be submitted for Mycobacterium tuberculosis amplification. Only specimens that are AFB stain positive are appropriate for testing. An AFB culture and smear should be ordered in conjunction with this request. This test is specific for, but does not differentiate between, members of the M. tuberculosis complex, i.e. M. tuberculosis, M. bovis BCG, M. africanum and M. micrati. A negative test does not exclude the possibility of isolating M. tuberculosis complex from the AFB culture.

• Test Request Form
• Specimen Containers
• Labeling
• Transport
• Results Reporting
• Rejection Criteria

Clinical Virology:

• Collection & Transportation of Specimens for Virus Isolation
• Collection Instructions by Specimen Type
• Rejection Conditions
• Virus Specific Specimen Guide
• Viral Culture Panels

Retrovirology
Parasitology:

• Collection Criteria

Antibiotic Susceptibility Testing:

• Antibiotic Assays
• Antibiotic Assays Monitoring
• Factors Affecting Monitoring of Selected Antibiotics

Culture Types & Guidelines:

• Blood Cultures
• Urine Cultures
• Respiratory Cultures
• Stool Cultures
• Routine Bacterial Cultures
• Miscellaneous Cultures
• Nose or Nasopharyngeal Swabs
• Throat Specimen
• Sputum Specimen
• Tracheal & Bronchial Aspirations
• Fungus
• Yeast
• Anaerobes
• Acid-Fast Bacillus

AFB Turnaround Times

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