Microbiology

General
Acid-Fast
Anaerobe
Blood
Exudates/Plus
Fungus
Hair
Ova and Parasites
Routine
Sensitivities
Skin
Sputum
Stool
Throat
Urine
Vag/Cervical (Genital)
Viral

General

Of all clinical laboratory procedures, valid microbiological test results probably are most dependent on proper specimen collection and transport techniques. The following procedures should be followed closely in order to provide a clinically relevant test result to aide the physician in diagnosis and therapy. The properly selected, collected and transported microbiological specimen ultimately will be of most benefit to the patient.

General Precautions

• When a "routine culture" is ordered, the laboratory will test the patient's specimen for all bacteria that are reasonably expected to cause infections in the part of the body from which the specimen was obtained. Anaerobic work-ups for appropriate sources must be ordered separately. If the specimens are to be tested either exclusively or additionally for other types of microorganisms (e.g., fungi or mycobacteria), you must specifically order separate cultures for them.
• Always indicate specimen source. With respect to lesions, it is most important for the laboratory to know first, the nature of the lesion, and second, the part of the body where it is located.
• Please provide the laboratory with whichever information you reasonably can give about the patient. Indicate the patient's name, sex and age at the top of the Test Request Form. A suspected diagnosis is helpful and, in some cases, absolutely essential. In addition, specify the date/time of specimen collection
• It is most desirable if specimens for culture are obtained before any antibiotic therapy is initiated. However, in cases where this is not feasible, please note which type of therapy the patient is receiving.
  • In collecting microbiological specimens, practice good aseptic technique.
  • If a wound or other lesion is being sampled, be particularly careful to obtain a "point" source, i.e., do not contaminate the sampling swab by touching it to surrounding tissues. Totally misleading results may be produced if this type of contamination occurs.
  • Do not touch the rims or interiors of specimen transport devices to any object other than the specimen or sampling swab and, in turn, be sure that the sampling swab touches nothing but the specimen site and the interior of the transport device.
• When using Culturette, follow the manufacturer's directions after the sampling swab has been reseated in order to saturate the swab with the medium. This aids in the preservation of the organisms you have collected. To ensure the best results, Culturette should be sent to the laboratory as soon as possible.
• Check the expiration dates on the various specimen collection devices before using. Do not use out-dated materials, and do not over-order dated supplies.
• Always label all specimens with the patient's name and other information as outlined in the Labeling of Samples information provided.

Top ^ Acid-Fast Culture and Acid-Fast Smear

• Sputum-An early morning specimen procured by deep coughing in order to bring sputum from as deep in the respiratory tract as possible is necessary. Patient should brush teeth and rinse mouth thoroughly two to three times with water (never mouthwash or other disinfectants) before collecting the specimen in a sterile sputum collection container. Attach the screw cap securely. A 5 to 10 mL volume is adequate for each specimen collected. Remember that saliva is not sputum. Do not pool sputum specimens. Twenty-four-hour specimens are not acceptable. Refrigerate the specimen until pick-up time.
  Make certain all specimens for TB culture are sent to us in securely capped, screw-cap containers to prevent danger of infecting others.
• Gastric Lavage-Submit in a sterile screw-capped sputum collection tube. Refrigerate until pick-up. Send specimen to lab as soon as possible.
• Urine-Early morning midstream specimen in sterile screw-capped sputum collection tubes. Twenty-four-hour pooled urines are not acceptable. Refrigerate until pick-up.
• Bronchial Washings-Submit in a sterile collection tube. Refrigerate until pick-up.
• Tissue-Submit in a sterile screw-capped collection tube with a small amount of sterile saline. Refrigerate until pick-up.
• Spinal Fluid-Submit in a sterile screw-capped spinal fluid tube. Never refrigerate this specimen if a routine bacterial culture also is ordered. Leave at room temperature.
• Fluids (pleural, exudates, thoracentesis, joint, etc.)-Submit in a sterile screw-capped collection tube. Specimen can be anti-coagulated with citrate or heparin, if necessary. Refrigerate until pick-up.

Top ^ Anaerobe Cultures

The following are examples of clinical materials that ordinarily are acceptable for culture of anaerobic bacteria.

• Aspirated pus
• Tissue (biopsy, surgical, autopsy)
• Body fluids (cerebrospinal, pleural, paracentesis, pericardial, synovial)
• Transtracheal aspirates
• Direct lung aspirates
• Sulfur granules for patients with suspected Actinomycosis

Specimens not cultured for anaerobic bacteria include

• Throat or nasal/pharyngeal swabs
• Gingival swabs
• Expectorated sputum
• Bronchoscopic specimens not collected by a protective double lumen catheter
• Gastric contents, feces, rectal swabs, colocutaneous fistulae and colostomy stomata
• Surface material from decubitus ulcers, swab samples of other surfaces and sinus tracts
• Materials adjacent to skin or mucous membranes other than the above that have not been properly decontaminated
• Voided urine, cath urine
• Vaginal or cervical swabs

Transport body fluids and aspirates aseptically collected through disinfected sites in a syringe capped with a black rubber stopper or an anaerobic transport vial. Expel all air from the syringe before transfer to the vial. Always leave specimens for anaerobe culture at room temperature.

Top ^ Blood Cultures

We recommend at least three blood cultures spaced an hour apart be obtained from the patient by following the procedure below.

• The skin site is disinfected first by cleansing with 70 percent alcohol and, secondly, by applying 2 percent iodine in concentric fashion to the venipuncture site. "Instant" antisepsis never occurs, and iodine should remain intact on the skin for at least one minute. The intended venipuncture site should not be touched unless the fingers used for palpation are similarly disinfected.
• Using one aerobic and one anaerobic blood culture bottle, remove the plastic bottle caps. Do not remove the rubber stoppers from the bottles at any time.
• Disinfect the rubber bottle stoppers by cleansing with 70 percent alcohol and allow to dry.
• Using a sterile syringe and needle or butterfly adapter, draw 20 mL of blood.
• Introduce 10 mL of blood into each bottle culture by passing the needle through the rubber stoppers. Mix well to avoid clotting.
• Return the blood culture bottle set to the laboratory as soon as possible. Never refrigerate blood cultures.

We incubate blood cultures over a five-day period from the time incubation begins. Positive blood cultures are phoned to the primary caregiver.

We routinely perform antibiotic sensitivities on all aerobic bacterial isolates from blood cultures.

Top ^ Fungus Culture

• Sputum, urine, tissue, bronchial washings and fluid specimens may be collected and transported in the same manner as AFB specimens except that mouthwash rinses are advocated prior to sputum collections.
• Skin scrapings for isolation of dermatophytes. Cleanse the affected area with a damp sponge containing 70 percent alcohol. Allow the area to dry. Using a sterile scalpel blade, scrape off skin at the active border (periphery) of the lesion including some of the healthy area. Obtain as much material as possible. Place the specimen in a sterile petri dish or small clean envelope and refrigerate until pick-up. Do not place these specimens in an air-tight container or tube.
• Nail scrapings for onychomycosis. Cleanse the nail with 70 percent alcohol and allow to dry. Using a sterile scalpel blade, scrape the nail and discard the initial portion. Place the deeper scrapings in a sterile petri dish or small clean envelope and refrigerate until pick-up. Do not place these specimens in an air-tight container or tube.
• Hair. Pluck out the affected hairs and place in a sterile petri dish or small clean envelope. The use of hair oils, dressings and some shampoos may interfere with fungus isolation and should be avoided for a few days preceding specimen collection. Use of Wood's Lamp frequently is useful in selecting infected hairs.
• Exudates and pus. Whenever possible, these specimens should be collected by aseptic aspiration with a sterile syringe through a disinfected site. Transfer the aspirate to a transport vial. Refrigerate until pick-up.
• Tissue (biopsy, surgical, autopsy). An excellent specimen for the isolation of fungi. Place into a sterile screw-capped container with sterile saline. Refrigerate until pick-up.

NOTE: All fungus cultures are held four weeks before reporting negative.

Top ^ Exam for Ova and Parasites

• Stool O & P-Samples are routinely screened for protozoan cysts, worm eggs and larvae.
• Three stool specimens should be collected, spaced every other day apart and sent to the lab as soon as obtained. It is imperative that the stool specimen be placed into "Parasafe" fixative vials immediately after collection to avoid disintegration of parasitic elements. Portions of the stool containing blood or mucus should be sampled and submitted. No barium at least one week prior to collection.
• Cellophane Tape Prep for Pinworm Examb-The Cellophane Tape prep is performed by pressing the sticky side of a piece of clear (not frosted) cellophane tape to several areas of the perianal region. It is most important to obtain this specimen a few hours after the person has retired (perhaps 10 or 11 p.m.) or the first thing in the morning before a bowel movement or bath. Press the tape to a clear glass slide and submit to the laboratory. A pinworm collection kit is available from the microbiology lab.

Note: Stool specimens are unacceptable for pinworm diagnosis since stools will contain eggs in only 10 to 15 percent of cases.

Top ^ Antibiotic Sensitivities

Sensitivities are routinely performed on the following organisms unless they occur as "normal" flora.

• Gram negative rods
• Staphylococcus aureus
• Group D Streptococcus (including enterococcus)
• Group B Streptococcus (non-respiratory)

Sensitivities are not performed on the following organisms.

• Diphtheroids
• Fungi (yeasts and molds)
• Lactobacillus species
• Nonpathogenic" Neisseria species (i.e., from throat and sputum cultures)
• Bacillus species
• "Normal" flora
• Anaerobic bacteria

Top ^ Routine Cultures From Skin Areas

This is the most abused culture specimen. The skin bears its own indigenous microflora; organisms normally are found there. Lesion sites contiguous with skin may contain these same organisms. Collection procedures from these sites must be stringent and culture results should be interpreted cautiously.

If the lesion is capable of yielding fluid material, the method of choice is to obtain a syringe aspirate. The healthy lesion margin carefully is disinfected with 70 percent alcohol, and a sterile needle is passed through this area into the lesion's interior.

If open wounds cannot be sampled in this way, then a swab specimen is taken. The surface of the open wound first should be cleansed mechanically with a non-antiseptic soap, sterile water or saline, and the lesion carefully sampled, avoiding adjacent skin areas. Transport at room temperature in a Culturette.

Anaerobe cultures are not acceptable.

Top ^ Routine Sputum Culture

Morning specimens resulting from overnight accumulation of secretion yield the best diagnostic results.

There are two methods used to obtain a sputum specimen: natural cough and the aerosol technique. To obtain satisfactory "deep cough" sputum by natural means, the patient is instructed to

• To brush his teeth and gargle with water,
• Inhale air to the full capacity of the lung (to breathe as deeply as possible), and
• Exhale air with an expulsive cough.

The alternative method employs an aerosol machine, which on the inhalation of a mixture of saline and glycol into the lungs, results in a forced cough. The forced cough is caused by produced moisture in the bronchial tree.

The sputum is collected in a sputum cup and sent to the laboratory immediately.

Top ^ Stool Culture

Presently the microbiology laboratory screens stool culture specimen for the presence of Shigella, Salmonella, Staph aureus and Campylobacter. Requests for additional pathogens (Yersinia, Enterohemorrhagic E. coli, etc) should be placed in the text comments.

Stools for culture should be submitted in a Culturette. Please write "stool" source on the requisition, and avoid the use of ambiguous terms such as rectal, anus, etc., if you want a culture screen for the enteric bacterial pathogens mentioned above.

Please note that stool specimens received in any fixative substance (e.g., formalin, Parasafe) cannot be cultured for pathogens.

Top ^ Throat Culture

For collection, the patient's head is tilted back, and the throat is well illuminated. The tongue is depressed so that the back of the throat clearly can be seen. A sterile cotton or Dacron swab is rubbed up and down the back of the throat and against any white or deeply reddened areas in the tonsilar area. The uvula, tongue and cheek are avoided. Place the swab into a Culturette and leave at room temperature until pick-up.

In the Routine Culture of the throat swab, an assessment is made of the bacterial and yeast flora isolated regarding its "normal" or "abnormal" nature to that site. Group A Streptococcus (Streptococcus pyogenes), the most commonly isolated significant pharyngeal bacterial pathogen, will be reported in any amount.

In the "Beta Strep Screen," Beta Hemolytic Streptococcus will be screened and reported in any amount.

Note that throat swabs for culture of Neisseria gonorrhoeae (gonococcal pharyngitis) must be immediately inoculated to a Martin-Lewis plate or into a Culturette and sent immediately to the lab.

Cultures of whooping cough (Bordetella pertussis) or Diphtheria (Corynebacterium diphtheriae) are performed by this laboratory. Phone the microbiology department if culture for these organisms is under consideration, as specific media are needed for their growth requirement.

Top ^ Urine Culture Sensitivities

We routinely perform sensitivities on clean catch urine isolates only if the colony count for that organism is equal to or greater than 10,000 col/mL.

Isolates from cath urine specimens routinely receive sensitivities.

Top ^ Genital Culture

Vaginal, Cervical and Urethral Culture
Usually, these are submitted as swab specimens from the affected area. Due to the abundance and complexity of the microbial flora indigenous to the vagina, cultures of specimens from this site generally should be limited to the isolation and identification of microorganisms associated with sexually transmitted diseases. Culture for any other diseases is not recommended. However, those specimens obtained at the time of pelvic surgery, those obtained by laparoscopy or culdocentesis and those obtained by needle aspiration of an abscess should be transferred to an anaerobic transport Culturette and cultured for aerobic and anaerobic bacteria.

Please note that anaerobe cultures are not performed on swab specimens of vaginal, cervical or urethral sites because of the usual indigenous anaerobic microflora of these areas.

Transport vaginal, cervical and urethral swab specimens in Culturette, and keep at room temperature prior to pick-up. Be sure to give the source of the specimen.

G.C. Culture (Neisseria gonorrhoeae)
Normally these are specimens from cervical, urethral, rectal or pharyngeal sites. These specimens may be collected in a Culturette or placed directly on a Martin-Lewis plate. The plate is inoculated by rolling the swab in a "Z" fashion across the media surface. Leave the plate at room temperature and take it immediately to the Microbiology Lab.

We strongly advise against the use of Gram stained smears to screen for gonorrhea, especially in the female patient. The indigenous flora of the vagina-and sometimes urethra-may contain bacteria resembling the gonococcus in Gram's stain (e.g., Moraxella, Acinetobacter, Neisseria lactamica, Neisseria meningitides, Branhamella).

The specific G.C. culture or DNA Probe for G.C. are the only certain means of diagnosis of this disease.

Top ^ Viral Specimen Collection

Collection of Specimens for Direct Immunofluorescent Antibody to Detect Viral Antigen
High quality specimens ensure high quality results. The following guidelines should be followed in collecting specimens for FA testing.

• The specimen should be collected as early as possible in the course of a patient's illness.
• Specimens must contain cellular material from the site of infection.
• Deliver specimens immediately to Laboratory Central Receiving or the Virology Laboratory on wet ice. Keep specimens cold before transport.
• Do not place specimens in transport media.
• Always accompany specimen with virology requisition. Particular DFA test (virus or viruses) must be checked or test cannot be performed.

Most FA tests can be completed in two to four hours if specimens are received in the morning hours. For same-day results, specimens must be received before 2:00 p.m. (with the exception of already prepared slides). Specimens received on weekends and holidays will be completed if time permits. All positive DFA results are called to the requesting physician/office.

Slide preparations

• Conjunctival swab (for suspected adenovirus, herpes simplex or varicella infection)-Use a pre-moistened (sterile saline) Dacron-tipped swab to swab the conjunctiva. Avoid mucus, if possible. Roll swab on a clean glass slide in two pea-sized circles. Allow slide to air dry. Label with the patient's name.
• Lesions or vesicles (for suspected herpes simplex or varicella-zoster)-Attempts to recover virus from crusted lesions are a waste of time. Use only Dacron swabs with plastic shafts. Unroof the blister and swab the base and roll swab on clean glass slide in two pea-sized circles (three circles for HSV and varicella-zoster testing). Or, after unroofing the blister, press a clean glass slide on the base of the lesion firmly on two spots. Allow slide to air dry. Label with patient's name.

Fluid or tissue collection

• Autopsy/biopsy tissue-Place tissue in sterile container; do not allow tissue to dry. Send as is or a touch prep or tissue impression can be made by rolling tissue with pressure on clean glass slide. Allow slide to air dry. Label with patient's name.
• Nasal (nasopharyngeal) washing-Two methods can be used: Luken's trap or syringe and butterfly technique.
  • The Luken's trap method requires normal saline to be injected into each nostril and immediately aspirated into the trap.
  • Utilizing the syringe technique, a butterfly is attached to a sterile syringe and 1-3 mL of sterile saline is drawn into the syringe. The needle and all but 3-4 cm of tubing are cut away. The remaining tubing is inserted into the nasopharynx, and 1-2 mL is injected and immediately reaspirated. Cap syringe with black, rubber stopper.
  • Do not place in viral transport media; send "as is" in sterile container. Be sure to obtain a "cloudy" specimen, since test cannot be interpreted if cells are not present. Send on wet ice as soon as possible (cells will breakdown within hours).
• Sputum-Collect in sterile container. Label with patient's name, identification number, date/time, source of specimen and identity of collector.

Collection of Specimens for Culturing of Viruses and Chlamydia

High quality specimens ensure high quality results. The following guidelines should be followed in collecting specimens for culturing (isolation) of viruses and Chlamydia.

• The specimen should be collected as early as possible in the course of a patient's illness.
• Viral specimens for culture should be placed in a viral transport medium (VTM), which is supplied by the Virology Laboratory. It is supplied frozen, in 2 mL quantities, in glass, black screw-cap vials. VTM should be kept frozen until ready to use. To thaw, rub between hands for two to three minutes.
• Chlamydia specimens for culture should be collected in Chlamydia transport medium. It is supplied as a clear liquid in plastic, conical centrifuge tubes with a blue cap; it must be stored refrigerated. Do not collect in viral transport media (these will be rejected). Do not collect with wooden swab (these will be rejected). Culture is considered the most sensitive and specific for diagnosing Chlamydia infections. This Chlamydia culture will detect all three species: C. trachomatis, C. psittaci and C. pneumoniae.
• Deliver specimens immediately to Laboratory Central Receiving or the Virology Laboratory on wet ice. Keep specimens cold before transport.
• Specimens must contain viable (living) infectious organisms; collection and transport are critical to virus and chlamydial viability. Respiratory viruses, especially RSV, are very labile.
• Always label specimen container with patient's name.
• Do not collect any viral or Chlamydia specimens in a Culturette (these will be rejected).
• " Only HSV will grow in one day, all other viruses will take two to 10 days for positive growth and result. All positive results are called to the requesting physician/office.

Specimen collection

• Cervical swab (for suspected virus: HSV, Chlamydia)-Use a speculum so that swabs may be collected properly with visualization of any lesions that may be present. Use only Dacron-tipped swabs. For Chlamydia, the endocervix and transitional epithelium must be sampled (removing excess mucus prior to swabbing). Vaginal swabs are not satisfactory for Chlamydia isolation. Place swab in viral or chlamydial transport medium, twirl vigorously, drain and discard swab. (Swab may be left in media, but be sure to cap without leakage).
• Conjunctival swab/eye swab (for suspected virus: HSV, varicella-zoster, adenovirus, Chlamydia)-Use a premoistened, Dacron-tipped swab to collect conjunctival cells and secretions. Place swab in viral or Chlamydia transport medium, twirl vigorously, drain and discard swab. (Swab may be left in media, but be sure to cap without leakage).
• CSF (for suspected virus: HSV, enterovirus)-Obtain 2 mL as for bacteriologic culture. Do not dilute or put in transport media. Place in sterile, tightly capped tube.
• Nasal wash-Two methods can be used: Luken's trap or syringe and butterfly techniques. For the trap method, normal saline is injected into each nostril and immediately aspirated into the trap. Utilizing the syringe technique, a butterfly is attached to a sterile syringe and 1-3 mL of sterile saline drawn into the syringe. The needle and all but 3-4 cm of tubing are cut away. The remaining tubing is inserted into the nasopharynx and 1-2 mL injected and immediately reaspirated. Be sure to obtain a "cloudy" specimen to ensure a high yield of virus. For culture specimens only, place washings in viral transport media or Chlamydia transport media.
• Nasopharyngeal swab-Swab each nostril, preferably gathering as much secretion as possible before removing swab, then rotate swab in viral or Chlamydia transport medium. Using a fresh swab, collect a throat swab and agitate it in the same vial of transport medium. Drain and discard swab.
• Rectal swab (for suspected virus: enterovirus, adenovirus, HSV)-Premoisten swab with sterile saline, place deeply into the rectum and twirl. Place swab immediately in viral transport medium, swirl vigorously, drain and discard swab or leave swab in media. Be sure to obtain sufficient fecal material on swab.
• Skin lesions (for suspected virus: HSV, varicella-zoster, enterovirus)-If vesicles are present, aspirate the vesicular fluid with a 25-gauge needle on a tuberculin syringe. Place this fluid in the viral transport medium. Then unroof the blister and swab the base of the lesion, and place this in the vial with vesicular fluid. Attempts to recover virus from crusted lesions are a waste of time.
• Stool (for suspected virus: rotavirus, enterovirus)-Requires small amount (0.5 g) of freshly passed stool. Place in plastic specimen container with tight-fitting lid.
• Throat-The posterior pharynx and any visible lesion should be vigorously swabbed with a Dacron-tipped applicator swab. Place the swab in transport media, swirl vigorously, drain and discard swab or leave swab in media.
• Urethral swab (for suspected virus: HSV, Chlamydia)-To prevent any organisms from being washed away, it is preferable to instruct the patient not to urinate for one hour prior to sampling. Insert small wire shaft swab 2-4 cm into urethra, gently rotate, remove and place in transport medium. Swirl vigorously, drain and discard swab.
• Urine-Freshly passed first morning void is the preferred specimen for CMV. Random samples are adequate for infants. Take great care in collecting specimens as aseptically as possible; collect in a sterile cup and cap tightly to prevent leakage.
• Autopsy/biopsy tissue-Collect a 1 cm3 tissue specimen with a sterile instrument. Place tissue in sterile container and flood with sterile saline or place in viral or Chlamydia transport media.

Collection of Specimens for DNA Probe Hybridization Testing for Chlamydia trachomatis and/or Neisseria gonorrhoeae

General

• Collection of a single swab can allow testing for both organisms. Because detection is at the genetic level, organisms need not be viable for a positive result.
• This assay eliminates problems with media storage and specimen transport encountered with culture. Once collected, specimens are stable at room temperature for seven days.
• The C. trachomatis probe assay is FDA approved for use on urogenital and conjunctival specimens only. Culture still will be available to detect all three Chlamydia species and must be used for non-genital sites (i.e. rectal, respiratory) and in cases of rape or child abuse.
• The N. gonorrhoeae probe assay is FDA approved for use on urogenital sites only. Culture still will be available through the Microbiology Laboratory.
• Chlamydia infection often is asymptomatic in both sexes. Gonorrhea is asymptomatic in 60 to 80 percent of women infected. There is a high incidence of co-infection with Chlamydia and Gonorrhea (25 to 50 percent in heterosexual men and women), therefore it is recommended that when one of these infections is suspect, the testing for both organisms should be considered.
• Specimens collected with this system cannot be used for culture. Only swabs supplied with the Gen-Probe specimen collection system should be used for specimen collection.
• Specimen collection kits (with separate collection kits for males [blue] and females [pink]) will be supplied by the Virology Laboratory

Collection of Cervical Specimens

• Remove excess mucus from cervical os and surrounding mucosa using one of the swabs provided. Discard the cleaning swab.
• Insert second swab from collection kit 1-1_ cm into endocervical canal.
• Withdraw swab carefully; avoid any contact with vaginal mucosa.
• See Preparation for Transport below.

Collection of Urethral Specimens:

• Patient should not have urinated for at least one hour prior to sample collection.
• Insert swab from collection kit 2-4 cm into urethra.
• Once inserted, gently rotate swab at least one full rotation using sufficient pressure to ensure swab comes into contact with all urethral surfaces. Allow swab to remain inserted for two to three seconds.
• Withdraw swab.
• See Preparation for Transport below.

Preparation for Transport:

• Insert one swab (collection swab only) into the Gen-Probe transport tube.
• Snap off shaft at score line or cut shaft to fit tube.
• For best results, transport tube to the laboratory at 2ﾡC-25ﾡC. Store at 2ﾡC-25ﾡC and test within seven days of collection.

Top ^