Specimen Collection

Basic Principles of Specimen Collection

To ensure appropriate specimen management:

If possible, collect the specimen in the acute phase of the infection and before antibiotics are administered.

Select the correct anatomic site for collection of the specimen.

Collect the specimen using the proper technique and supplies with minimal

contamination from normal flora. Collect the appropriate quantity of

specimen. Package the specimen in a container

designed to maintain the viability of the organisms and avoid hazards that result from leakage.

Label the specimen accurately with the specific anatomic site and the patient information.

Transport the specimen to the laboratory promptly or make provisions to store the specimen in an environment that will not degrade the suspected organisms.

Consideration in Specimen Collection: Specimen should be collected in sterile

containers. Swabs are not recommended for

collection because:

a. they do not provide sufficient quantity

b. they can easily be contaminated

c. can become dried out, leading to a loss of organisms.

Swabs are appropriate if specimens are from:

a. external ear c. upper respiratory tract

b. eye d. genital tract

Patients-Collected SpecimensThe specimens commonly obtained by the patient are:a. urine c. stoolb. sputum

Things to Consider in Collecting Urine Sample:a. It must be a clean-catch midstream urine

specimen.

b. First morning specimen is preferred

c. The patient collects this specimen following cleansing of the external genitalia.

Things to Consider in Collecting Sputum Sample:

a. The first early morning specimen is preferred.

b. The patient should rinse the mouth with water and expectorate with the aid of

a deep cough directly into a sterile container.

c. Patients with dentures should remove the dentures first.

2 Methods of Collecting Stool Specimen:

a. rectal swab b. stool excreted

Rectal Swab: 3 specimens should be collected, once a day for 3 days.

Stool Excreted: stool should never be taken from the toilet and should not be contaminated with urine.

SPECIMENPATIENT

PREPARATIONCONTAINER/MINIMUM QUANTITY

Blood Culture Disinfect skin w/ alcohol

Blood culture media set (aerobic & anaerobic bottles) or vacutainer tube w/ SPS(adults: 20 ml. per set; children: 5-10 ml. per set).

Ear: Outer Ear Remove debris or crust from ear canal with saline moistened swab; rotate swab in outer canal.

Swab transport system

Eye: Conjunctiva Sample both eyes; use separate swabs moistened with sterile saline

Swab transport system

SPECIMEN COLLECTION GUIDELINES

Feces Collect directly into container, avoid contamination w/ urine

Clean, leak-proof container or enteric transport system

Fungal Scrapings Wipe nails or skin w/ alcohol;Hair: 10-12 hairs w/ shaft intactNails: clip affected areaSkin: scrape skin at outer edge of lesion

Clean, screw-cap container

Genitalia: Vagina Remove mucus before collection; do not use lubricant on speculum, swab endocervical canal or vaginal mucosa

Swab transport system or JEMBEC transport system

Lesion/ Wound/Abscess (Superficial)

Wipe area w/ sterile saline or alcohol, swab along outer edge.

Swab transport system

Respiratory tract: Lower sputum

Rinse mouth or gargle w/ water, instruct to cough deeply into container

Sterile, screw-cap container

Throat Swab posterior pharynx, tonsils and inflamed areas

Swab transport sytem

Urine: Clean-catch midstream

Clean external genitalia, begin voiding and after several ml. have passed, collect midstream w/o stopping flow of urine

Sterile, screw-cap container or Urine transport kit

Labelling & Requisitions:

Proper identification of each specimen

includes a label firmly attached to the

container with the following information:

Name / age Date of collection

Room number Time of collection

Physician’s name Laboratory number

Culture site

Labelling and requisitions will determine exactly what needs to be done.

Laboratory Safety

Laboratory personnel must adhere to

strict safety guidelines as they begin to work

w/ the patient’s specimen and must wear

protective clothing and specimens should be

opened only in a biological safety cabinet.

Preservation, Storage and Transport of Specimen

Specimen Storage:

Some specimens such as urine, stool,

sputum, swabs (not for anaerobes) can be

maintained at refrigerator temperature (40C)

Pathogens that are cold sensitive

maybe found in other specimens that should

be kept at room temperature.

Transport Media

Transport media contain substances that do not promote multiplication of microorganisms but ensure the preservation especially specimens collected by swab.

Ex: Stuart or Amie’s transport media

Cary-Blair transport media

Blood: Broth culture medium

Bacterial Cultivation

Cultivation – is the process of taking the

bacteria from the infection site and letting it

grow in an artificial environment called the

culture media.

3 Main Purposes of Bacterial Cultivation:

a. To grow and isolate all bacteria present in a clinical specimen.

b. To determine w/c of the bacteria that

grow are most likely to cause infection and

w/c are likely contaminants or colonizers.

c. To obtain sufficient growth of clinically

relevant bacteria to allow identification and

characterization

Nutritional Requirement - this include:

gases nitrogen

water sources for carbon

various ions energy

2 Phases of Culture Media:

a. liquid (broth) b. solid (agar)

In broth media, bacterial growth is

indicated by a change in the broth’s appearance from clear to turbid.

Solid media are made by adding agarose.

With appropriate incubation, bacterial cell inoculated onto the agar medium will proliferate to sufficient number (bacterial population – “colony”) that can be seen by the naked eye.

Classification of Media According to

Function / Use:

a. Enrichment Media – contain specific

nutrients required for the growth of

particular bacterial pathogens (fastidious

organisms) that may be present alone or w/

other bacteria in the specimen.

b. Selective Media – support the growth of

one type or group of microbes but not

another. This medium may contain

inhibitory substances such as antimicrobials,

dyes or alcohol.

Ex: Mac-Conkey agar for enteric gm (-)

bacilli.

c. Differential Media – employ some factors

that allow colonies of one bacterial species

or type to exhibit certain metabolic or culture characteristics that can be used to distinguish them from other bacteria growing on the same agar plate.

Ex: Mac-Conkey agar – inhibits gm(+) and differentiates gm(-) bacilli on the basis of lactose fermentation.d. Supportive Media – contain nutrients that

support growth of most non-fastidious

organisms w/o giving any particular

organism a growth advantage.

CULTURE MEDIA & ITS USEMEDIUM 10 PURPOSE

Blood Agar Cultivation of fastidious microorganisms, determination of hemolytic reactions

Chocolate Agar Cultivation of Haemophilus spp. and pathogenic Neisseria spp.

Eosin Methylene Blue (EMB)

Isolation and differentiation of lactose-fermenting and non lactose-fermenting enteric bacilli

MEDIUM 10 PURPOSE

Mac-Conkey Agar Isolation and differentiation of lactose-fermenting and non lactose-fermenting enteric bacilli

Salmonella-Shigella (SS) Agar

Selective for Salmonella and Shigella spp.

Selenite broth Enrichment of isolation of Salmonella spp.

Thayer-Martin Agar Selective for N. gonorrhea & N. meningitidis

Thioglycollate broth Supports growth of anaerobes, aerobes, microaerophillic, and fastidious organisms

MEDIUM 10 PURPOSE

Thiosulfate Citrate Bile Salts (TCBS) agar

Selective and differential for vibrios

Trypticase Soy Broth (TSB)

Enrichment broth used for subculturing various bacteria from primary agar plates.

Xylose Lysine Desoxycholate (XLD) agar

Isolation and differentiation of Salmonella and Shigella spp. from other gm(-)enteric bacilli

Brain-Heart Infusion (BHI) – is a

nutritionally rich medium used to grow

various microorganisms, either as a broth or

as an agar, with or w/o added blood.

Chocolate agar – is essentially the same as

blood agar except that during preparation

the red blood cells are lysed when added to

molten agar base. This lysis releases intra-

Cellular nutrients such as hemoglobin, hemin

and the coenzyme nicotinamide adenine

dinucleotide into the agar for utilization by

fastidious bacteria.

The most common bacterial pathogens

that require this enriched medium for growth

include Neisseria gonorrhea, the causative

agent of gonorrhea, and Haemophilus spp.

Mac-Conkey agar – is the most frequently

used primary selective and differential agar.

This medium contains crystal violet dye to

inhibit the growth of gm(+) bacteria and

fungi, and allows many types of gm(-)

bacilli to grow.

With Neutral red indicator – resulting to

pink to red colonies.

Sheep Blood agar – this medium supports all but the most fastidious clinically significant bacteria.

Certain bacteria produce extracellular enzymes that lyse red blood cells in the agar known as hemolysis.

Types of Hemolysis produced by bacteria:a. alpha hemolysis c. gamma or no b. beta hemolysis hemolysis

Thayer-Martin agar – is an enrichment and

selective medium for the isolation of

Neisseria gonorrhea and Neisseria

Meningitidis. The enrichment portion of the

medium is the basal components and the

chocolatized blood, while the addition of

antibiotics provides a selective capacity.

The antibiotics include colistin to inhibit

other gm(-) bacteria, vancomycin to inhibit

gm(+) bacteria, and nystatin to inhibit yeast.

The antimicrobial trimethoprim is also

added to inhibit Proteus spp., w/c tend to

swarm over the agar surface and mask the

detection of individual colonies of the two

pathogenic Neisseria spp.

Thioglycollate broth – contains many nutrient factors, including casein, yeast and beef extracts, and vitamins to enhance the growth of most medically important bacteria. This agar supplement and the presence of thioglycolic acid, w/c acts as a reducing agent to create an anaerobic environment deeper in the tube, allows anaerobic bacteria to grow.

Xylose-Lysine-Desoxycholate (XLD) agar –a phenol red indicator in the medium detects increased acidity from carbohydrate fermentation. Because of decarboxylation of lysine, the pH (culture environment) increases thus causing the pH indicator to turn red. These colonies often exhibit a black center that results from Salmonella

spp. Producing H2S.

Preparation of Artificial Media:Generally, media are reconstituted by

dissolving a specified amount of media powder, w/c usually contains all necessary components in water. Boiling is often required to dissolve the powder, but specific manufacturer’s instructions printed in media package inserts should be followed exactly. Most media require sterilization so that only

bacteria from patient specimens will grow

and not those that are contaminants from

water or the powdered media.

Media Sterilization

The timing of autoclave sterilization

should start from the moment the

temperature reaches 1210C and usually

requires a minimum of 15 mins.

Streaking an Agar Plate for Isolation: The 3 Sector Method

1. Lift the lid of the petri plate just enough

to insert the inoculating loop. Keeping the

loop parallel w/ the agar surface and starting

at 12:00 position, zigzag the loop back &

forth across the agar surface until you have

covered approximately 1/3 of the plate.

2. Sterilize the inoculating loop in your

gas burner and stick the loop into the agar at

the edge of the agar to cool.

3. Now rotate the plate 450 counterclock –

wise so that the area you just streaked at the

12:00 position is now at the 9:00 position.

4. Take the sterile inoculating loop and

drag it through “area 1” two-three times

to pick up some of the bacteria already on

the plate and spread them over half the

remaining plate. Do not touch “area 1” after

the first 2 or three drags.

5. Sterilize the inoculating loop in your

gas burner and stick the loop into the agar at

the edge of the agar to cool.

6. Now rotate the plate 450 counterclock-wise

so that the area 2 that you just streaked at the

12:00 position is now at the 9:00 position.

7. Take the sterile inoculating loop and

drag it through “area 2” two-three times to

pick up some of the bacteria already on the

plate and spread them over most of the

remainder of the plate. Do not touch “area

2” after the first two or three drags and be

careful not to drag the loop into “area 1.”

8. Close the lid of the plate and sterilize

the inoculating loop.

Incubation:Incubation conditions must be

considered when a medium is inoculated. Most bacteria cultures are incubated at 350C to 370C.

Classification of Bacteria as to Oxygen Requirement:

a. Aerobes – bacteria that grow in ambient air or in the presence of an oxygen.

b. Anaerobes – bacteria that grow in the absence of an oxygen.

c. Microaerophiles – bacteria that grow w/ reduced oxygen and increased carbon dioxide.

d. Capnophiles – bacteria that require an increased concentration of carbon dioxide (ex. by a candle jar, carbon dioxide incubator).

MICROORGANISM CULTURAL APPEARANCE

Staphylococci Small to medium, opaque, smooth, slightly raised, translucent, creamy yellow

Streptococci Grayish white, small or minute, matte or glossy, flat.

B. Anthracis Medium-large, gray, flat, irregular w/ swirling projections (“Medusa head”).