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Collection and Transportation of Microbiological

The laboratory diagnosis of an infectious disease begins with the collection of a clinical specimen. Proper collection of an appropriate clinical specimen and transported in the right environment to the laboratory is the first step in obtaining an accurate laboratory diagnosis of an infectious disease.

Basic requirements before sending specimens for culture

Following measures should be taken before submitting specimens for culture to the clinical laboratory.

  1. Collect the specimen before the administration of antimicrobial agents

  2. The specimen should be collected carefully to prevent the contamination of the specimen with superficial organisms or normal flora of the body

  3. Make sure that the container is not contaminated, and is leak proof.

  4. Try to maintain strict aseptic techniques throughout the procedure.

  5. Make certain that the specimen is representative of the infectious process (e.g. early morning quality sputum sample) and is adequate in quantity.

  6. Label the container appropriately and complete the requisition form.

  7. The specimen should be transported immediately to the laboratory.

  8. 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 organisms is essential for the optimal and appropriate processing of the specimen.

BLOOD CULTURE:

  1. After placing a tourniquet on the patient's arm, select a suitable vein.

  2. Cleanse the area with the 2% iodine followed by 70% alcohol, in circular motion from the centre to outside.

  3. Without touching the site of the venipuncture with the fingers, draw 10-20mL of blood into a sterile syringe. If other blood tests are required than first take the blood culture from aseptic site.

  4. First clean the blood culture bottle stoppers with alcohol than inoculate 5-10mL of the specimen into each of two culture bottles Gently invert the bottles several times to insure thorough mixture of the contents.

POINTS THAT SHOULD BE COMMUNICATED TO THE CLINICIANS BEFPRE BLOOD CULUTRE COLLECTION

  1. Collect blood during the early stages of disease, during peak of fever since the number of bacteria in blood is higher in the acute and early stages of disease.

  2. Blood cultures should be sent prior to antibiotic administration.

  3. Three blood cultures sets are recommended at one hour intervals to obtain 99% positivity.

Small children usually have higher number of bacteria in their blood as compared to adults and hence lesser volume (3-8 mL) is required for culture.

CSF:

Examination of CSF is an essential step in the diagnosis of any patient with evidence of meningeal irritation or affected cerebrum. Following important precautions need to be taken for CSF collection and transportation.

  1. Collect CSF in a screw capped sterile container and not in an injection vial with cotton plug.

  2. Almost 3-10 ml of CSF should be collected for biochemical, immunological and microscopic examination.

  3. Do not delay transport and laboratory investigations.

  4. Transport in a transport medium if delay in processing is unavoidable.

  5. CSF is a precious specimen, handle it carefully and economically. It may not be possible to get a repeat specimen.

Perform physical inspection immediately after collection and indicate findings on laboratory requisition form.

SPUTUM:

  1. Select a good wide-mouthed sputum sterile container, which is preferably disposable, made of clear thin plastic, unbreakable and leak proof material.

  2. Three early morning sputum specimens are recommended for diagnosis of tuberculosis

  3. Explain to the patient to rinse his/her mouth with plain water before bringing up the sputum.

  4. Instruct the patient to inhale deeply 2-3 times, cough up deeply from the chest and spit in the sputum container by bringing it closer to the mouth.

  5. Make sure the sputum sample is of good quality. A good sputum sample is thick, purulent and sufficient in amount (2-3 ml).

  6. Do not submit saliva to the laboratory as it would lead to erroneous results.

Urine:

The lower part of the urethra and the genitalia are normally colonized by bacteria, therefore proper aseptic techniques should be used to prevent contamination of urine with the perianal flora. Therefore for microbiological examination (c\s) urine must be collected as a "clean catch-mid-stream" specimen. Early morning specimen is recommended usually however if this is not possible, then specimen should be collected two hours after last micturation. Urine specimens should be transported to the laboratory within one hour for bacteriological examination. If delay is expected in transportation collect specimen in 1 % Boric Acid and containers brought to laboratory with in 4-6 hours. Transportation should be in cooling box (2-80c) except when transport time is very short. Indicate clearly whether the specimen is from a clean catch or a catheter.

 Male

  1. If not circumcised, draw back the fore skin.

  2. Discard initial few ml of the urine.

  3. Collect the mid-portion of the urine into container, and pass the excess in the toilet.

Female

  1. Wash the area around the urethra by means of running water.

  2. Squat over the toilet and separate the labia with one hand.

  3. Discard initial few ml in the toilet and collect the mid-portion of urine in the container.

For Indwelling Catheter urine specimen:

  1. Clean the catheter collection port with 70% alcohol wipe.

  2. Using sterile technique, puncture the collection port with a needle attached to the syringe.

  3. Aspirate the urine and place in sterile container (green top container).

Do not collect urine from the collection bag.

Wound Specimens & Aspirates:

  • Aspirated specimens, fluids, tissues are good quality specimens that are preferable than specimens collected on swabs.

  • Gram stain is not recommended from swab specimens.

  • Mention special instructions or specific tests to be done on requisition slip.(e.g. for fungal culture or AFB culture or Nocardia or any other fastidious bacteria)

  • Try to follow the universal precautions for specimen collection if collecting with swab.

  • Swabs are unacceptable for anaerobic culture and TB cultures.

  • Try to transport the aspirate as early as possible to the laboratory in order to prevent contamination and also for maximum yield of aerobic as well as anaerobic bacteria.

Stool Specimens:

  • For parasitic examination, fresh stool specimen is recommended; delayed transportations will cease the mobility of trophozoites.

  • Specimen should be collected in a clean wide mouthed plastic bottle. A wooden stick to transfer the specimen should be provided.

  • For bacterial culture also, fresh stool must be submitted, if delay in transportation is expected to be more than 2 hours, specimen should be submitted in Cary Blair Medium.

Cellophane Tape method for detection of pin worm ova (Enterobius vermicularis)

Procedure:

  1. Cut off a piece of cellophane tape (10cm long) and place one end on the microscopy slide.

  2. Wear gloves.

  3. Place the microscopy slide with the tape partially attached to it on a wooden depressor and fold the tape over the end of the depressor to expose the sticky surface.

  4. With one hand press the buttocks apart to expose to perianal region.

  5. Press the sticky surface against several areas of the perianal skin.

  6. The sticky surface is then placed with the sticky side down on to the slide

  7. Label the specimen and sent to the lab.

  8. No special requirements is required for transportation or storage.

Reference:

  1. Henry D Isenberg. Clinical Microbiology Procedure Handbook, 2nd ed. Vol. 1 ASM press

 

FUNGAL INFECTION OF SKIN, NAIL AND HAIR

How to collect specimen?

The dermatophytes are distinct group of fungi that infect the skin, hair and nails of human and animals, producing a variety of cutaneous infection commonly called as “RING WORM”.

Although over 30 species of dermatophyes are described the following six cause the majority of human dermatophytes infection.

 

GENUS

SPECIES

1) Epidermophyton

a) Epidermophyton flocossum

2)Microsporum

  • Microsporum canis.

  • Microsporum gypseum

3) Trichophyton

  1. Trichophyton entagrophytes

  2. Trichophyton rubrum.

  3. Trichophyton tonnsurans

Specimen Collection for diagnosis of cutaneous infections:

Suitable specimen selection, proper collection and rapid transport must be performed to ensure the successful isolation of the fungal pathogen. If there is any question about how to select, collect or transport a specimen the clinician should contact the microbiologist for assistance.

General considerations:

  1. Collect specimens aseptically in sterile leak proof containers.

  2. Deliver the specimens to laboratory within 2 hours.

  3. Rapid transport is essential to ensure the survival and to prevent the over growth by bacteria.

  4. An optimum amount of specimen should be sent to laboratory and inadequate specimen might lead to false negative results.

 

 
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