Selection, Collection, and Submission of Samples

Bacteriology and Mycology Submission Guidelines

Selection, Collection and Shipping of Specimens

The success and ultimate value of examining clinical specimens bacteriologically are influenced initially by the care exercised in the selection, collection and shipment of the specimens. Specimens selected should be those likely to yield the causative agent(s) and efforts should be made to avoid contamination with organisms from the surrounding environment.

Delivery of Specimens

Personal delivery or overnight shipment by commercial carrier including: the U.S. Post Office, UPS, Air Express, Federal Express, etc. is acceptable. The specimens should be shipped in accordance with IATA Packing Instruction 650 and with adequate amounts of ice packs to prevent deterioration. Questions regarding optimal specimen collection or shipping should be directed to KSVDL Client Services at 785-532-5650 or toll free 1-866-512-5650.

Bacterial Isolation Specimens

Tissues and organs—Suitable portions of each specimen should be placed in individual polyethylene bags or other suitable leakproof container. Portions of the intestine (tied off if appropriate) should be packaged separately.
Swabs—Swabs may be the preferred way for submitting specimens from nasal passages, pharynx, tonsil, eye, ear, skin, abscesses, vagina, cervix, etc. Drying of specimens after collection and during shipment must be avoided by using a suitable transport medium. Swabs in transport medium are available commercially.
Feces—Preferably, fecal specimens should be obtained directly from the rectum in a manner that avoids contamination, placed in a leakproof container, and immediately shipped to the laboratory. Because of likely contamination, submission of fresh droppings should be avoided if possible.
Milk— See section on milk culture for specific sample guidelines
Urine—Urine must be collected aseptically and refrigerated immediately. Free catch urine samples should be avoided if possible to avoid contamination. Better recovery of organisms is achieved from 1-5 ml of urine. Urine swabs are not recommended.
Brain— Place entire brain in a polyethylene bag or other leakproof container.
Equine cervical or uterine swabs—Sterile swabs with long handles, referred to as “disposable guarded culture instruments” are available commercially. These instruments do not contain a transport media and if there is going to be a time delay, a transport media should be used.

Disease Requiring Special Consideration
Clearly label all boxes, bags and submission forms from primates and those that are anthrax, plague, tuleremia, coccoides, or foreign disease suspects to avoid exposure to personnel

Anthrax—Cotton swabs are soaked in extruded blood or blood taken from a superficial ear vein in acute or paracute cases. In swine, swabs should be taken from exudates and cut surfaces of hemorrhagic lymph nodes.
Blackleg, malignant edema, etc.—Submission of fresh affected tissues is absolutely essential since Clostridia from the gut rapidly invades tissues after death. Two (2) impression smears on glass slides should be included with the submission.
Clostridial enterotoxemia—Several ounces of fresh intestinal contents or a tied-off section of affected intestine should be submitted to the laboratory as soon as possible.
Campylobacteriosis—Bovine and ovine—Semen, preputial washings, fetal stomach contents, and cervical mucus should be obtained as aseptically as possible and delivered to the laboratory under refrigeration within five to six hours of collection. Alternatively, specimens may be frozen as soon as collected with dry ice and shipped in an insulated container with an adequate amount of dry ice.
Brucellosis—Placenta, stomach content or aborted fetus, mammary lymph nodes, milk samples are the preferred specimens for the isolation of Brucella organisms. Specimens should be shipped to the diagnostic laboratory under refrigerated conditions.
Listeriosis—Submit brain stem sample and indicate Listeria is suspected.

Fungal Isolation Specimens

Fungal cultures are checked weekly for growth, but may take up to 4 weeks for slow growing isolates or before they are reported as negative. It is very important to obtain a proper specimen. General guidelines include using aseptic collection techniques, collection from sites representative of the disease process, collection before antifungal therapy, and collection of an adequate volume of material.

Superficial mycoses—Hair and skin scrapings from the edge of active lesions are preferred for the isolation of dermatophytes. Submit in a small paper envelope. Saprophytic fungi will frequently proliferate rapidly and overgrow ringworm fungi in a sealed container.
Deep-seated mycoses—Tissues and swabs as for bacteriology.

Blood Culture (Brucella Canis) Submission Guidelines

Contamination of blood cultures can lead to a false negative test result. To avoid contamination during sampling and handling of Brucella canis specimens, the following method should be followed:

Animals should be shaved in blood draw area and cleaned with an alcohol wipe
Blood should be drawn using a vacutainer holder and a BD Hemogard* sodium citrate tube. Tubes should never be opened by removing the cap. A separate sterile needle should be used for each animal.
*Hemogard tubes limit contamination during laboratory processing. The bacteriology laboratory can provide these tubes for a nominal fee.
Tubes containing other preservatives (EDTA, heparin) can not be used for B. canis culture.
Tubes should be labeled with sample number and unique animal identifier. This information should match the submission paperwork.
Tubes should be placed on ice packs and submitted to the KSVDL immediately using overnight shipping.

Anaerobic Culture Specimens

If the specimen is collected by needle aspiration, all the air must be expelled from the barrel of the syringe prior to collection. The contents then must be transferred to a sterile glass tube. Be sure to completely fill the tube, so as to leave no oxygen, which might be toxic to the anaerobes during transport. There are several commercial anaerobic systems that are used to successfully collect and transport anaerobic specimens to the laboratory. Unless tissue specimens can be delivered promptly to the laboratory or placed in an oxygen-free container, or are small enough to be placed in one of the commercial systems, culturing this type of specimen for anaerobes rarely provides useful information. Clinical conditions that are suggestive of anaerobic infections:

Foul-smelling discharges
Deep infections from penetration of cutaneous or mucosal surfaces
Necrotic tissue, gangrene, pseudomembrane formation
Gas in tissue or exudate
Endocarditis with a negative blood culture for aerobic bacteria
Infection associated with malignancy or other disease causing tissue destruction and impaired circulation
Bite wounds
Deep abscesses
Septic pleuritis
Aspiration pneumonia
Fractures associated with trauma to soft tissue
Infections following surgery of the gastrointestinal tract
Septic processes such as pyometra

Specimens that are suitable for Anaerobic Culture:

Normally sterile body fluids, such as pleural, peritoneal, joint and cerebrospinal fluid, bile, etc.
Surgical specimens from sites that normally are sterile
Deep abscess contents taken aseptically
Blood, if collected properly
Aspirates from deep wounds
Specimens obtained by specialized procedures, such as transtracheal aspiration

Specimens that are not suitable for Anaerobic Culture:

Saliva
Vaginal or cervical specimens
Feces (unless looking for a particular Clostridial pathogen)
Tracheal swabs
Naso-tracheal aspirates
Colostomy or ileostomy effluents
Skin or superficial wound swabs
Voided or catheterized urine

Individual Animal Milk Culture

The KSVDL microbiology laboratory adheres to the policies set forth by the National Mastitis Council in the Laboratory Handbook on Bovine Mastits. As such, individual animal milk samples with more than 3 colony types will be reported as “Contaminated”. Common sources of contamination are: dirty teat ends, milk touching hands or fingers before entering tube, non-sterile tubes or inoculating needles, and excess alcohol on teat ends or hands.

The key to quality milk culturing is aseptic sample collection (see method below). Contaminated samples can lead to misdiagnosis, increased work, and confusion. If you have any questions on aseptic milk collection, please call the microbiology laboratory (785-532-4468) and we can assist you. Milk samples should be collected and shipped in sterile vials or tubes; Whirl-pak bags, Ziplocs, and other storage type bags are not acceptable as they are unsterile and leak during shipping. The microbiology laboratory is able to provide milk culturing supplies for a nominal fee.

Procedure:

Label tubes
The person taking samples should have clean, dry hands. Wearing latex gloves is recommended.
Clean excessively dirty teats with water and dry the udder
Predip
Dry teats
Wipe the teat ends with alcohol. Disinfect the teats farthest from you first.
Forestrip 2-3 times
While holding the collection tube at an angle, take the sample. Do not touch the teat end with the collection tube. Teats closest to you should be sampled first.
Store samples on ice. (If milk samples are to be cultured 24-48 hours after collection, samples should be frozen.)

Common sources of contamination are: dirty teat ends, milk touches hands or fingers before entering tube, non-sterile tubes or inoculating needles, and excess alcohol on teat ends or hands.

Bulk-Tank Milk Culture

Bulk tank milk (BTM) culturing can supply two important types of information; presence or absence of a bacterial group and identification of predominant bacterial groups. The more often BTM is sampled, the more useful the information. Extreme caution should be taken when interpreting results from a single BTM sample. Bulk tank milk cultures are not useful as indicators of mastitis prevalence in a herd. Bulk tank milk cultures can be valuable supplements to quarter milk samples, but never a substitute for determining infection incidence and prevalence based on quarter milk samples.

Procedure:

Agitate the bulk tank for 10-15 minutes prior to sampling.
Take the sample from the top of the tank with a sterile dipper or syringe. Do not sample from the outlet valve.
Transfer 10-15 mL of the sample to a sterile collection vial or tube. Do not send samples in whirl-pak or other non-sterile containers.
Freeze the sample immediately.
Pack frozen samples on ice and ship to the laboratory by overnight courier.
Wipe the teat ends with alcohol. Disinfect the teats farthest from you first.

Please call if you have any questions on bulk tank milk culturing procedures.

Mammalian Mycoplasma Specimen Submission Guidelines

Swabs- Moist swabs from deep within the nasal passage, tonsils, trachea, and bronchi (use pediatric swabs to reach into the bronchi); vulvar swab, conjunctival swab, joint swab.
Swabs should be placed in an appropriate transport media (Amies' or Stuart's). Do not use swabs with wooden shafts or media containing charcoal.
Other acceptable specimens- Milk, joint fluid, joint capsule, tracheal and bronchial washes, thoracic fluid, pleura, pericardium, peritoneal fluid, fresh tissue with lesions.

Note- Because Mycoplasma hyopneumoniae is difficult to culture, a species specific PCR is performed on all porcine Mycoplasma submissions.

Avian Mycoplasma Specimen Submission Guidelines

Cloacal/choanal swab, tissues with lesions including air sacs, sinuses, joints, tracheal swabs, tissue or fluid from swollen sinuses or joints.

Material:

Swabs should be placed in an appropriate transport media (Amies' or Stuart's). Do not use swabs with wooden shafts or media containing charcoal.
Fresh tissue: Place appropriate sample in a Whirl-pak
Fluid Sample: Submit approximately 1 ml in a screw-cap or red-top tube.
Milk: Samples submitted for routine bacteriology are acceptable for Mycoplasma culture. See milk culture section for collection / shipping guidelines.

Specimen Transport: Ship specimens to be cultured on frozen gelpacks. When culture setup must be delayed beyond 48 hours, samples should be stored in liquid nitrogen or at -70°C. Never freeze specimens at -20°C.

If PCR is requested, refrigeration is preferred, but not mandatory.