#sheet (12).

made by: marah marahleh

corrected by: wael abu-anzeh

date 27/10/2016

Basic principle of specimen collection

"This is going to be the last lecture in the first exam."

We talked previously about the sample collecting process, the right volume, the

methodology , quality of specimen which are all factors that affect the identification

process .

Specimen Collection Guidelines

Disinfect skin with alcohol and iodine, blood culture media set Blood culture

(aerobic and anaerobic, bottles) or vacutainer tube with SPS( sodium polyanethol

sulfonate) /adults, 20 ml per set; children 5 to 10 ml per set

ile, joint, pericardial, pleural), disinfect (Abdominal, amniotic, ascites, b Body fluids

skin before needle aspiration sterile, screw-cap tube , ≥1 ml

cap -Disinfect skin before aspiration, use sterile screw Cerebrospinal fluid

tube/bacteria ≥1 ml, fungi, ≥2 ml, AFB ≥2 ml, virus ≥1 ml

The volume should be 1 ml , less than one ml won't be accurate and the technician

have the right to reject the sample ( if any of the criteria of sample collecting isn't

met, the technician have the right to reject the sample otherwise it will give false

result ).

We also talked about blood culture , body fluid , CSF(cerebrospinal fluid ) and the

volume needed for every analysis if we're looking for bacteria , fungi , AFB( acid fast

bacilli) or viruses.

Ear

1. Inner ear: clean ear canal with mild soap, aspirate fluid, with needle if eardrum

intact; Or use swab if , eardrum ruptured, sterile, screw-cap tube or anaerobic

transport system

2. Outer ear : remove debris or crust from ear canal with saline moistened swab;

rotate swab in outer canal, swab transport system

Ear: 1) inner ear infection: more complicated, the physician collects the sample, the

needle used is the typical syringe not the 10 ml or 5 ml that is used for blood samples

if the eardrum is intact but if the eardrum is raptured we use ear swab we draw the

sample form the pus in small tubule.

We use anaerobic system if we are looking for anaerobic bacteria.

For anaerobic bacteria we don’t use the same method for aerobic bacteria if the

physician suspects that there is anaerobic infection mainly in the inner ear bacteria,

then the sample should be kept in an anaerobic transport system.

2) Outer ear: easily taken by the technician after removing the debris or crust from

the external ear canal with normal saline. The swab should be moisturized before the

sample is taken.

What is the meaning of transport media ?

Swab is not directly taken to the lab it is put in a transport media which is a tube that

contain agar to maintain the viability of the bacteria ( exact number ) during

transportation .

Sometime we need to transport the sample because the clinic where the sample is

taken is far away from the lab.

The agar used in the transport system is not nutritional and it is not used for the

multiplication of the bacteria.

The transport media contain suppressant media for normal flora and preservative to

maintain the viability of the bacteria .

Feces SAMPLES

- Collect directly into container, avoid contamination with urine, clean, leak proof

container or enteric transport system.

- A rectal swab can be submitted for bacterial culture but it must show feces. A

single specimen is not usually sufficient to exclude bacteria or parasites.

- if a bacterial infection is suspected, three specimens should be collected, one a

day for 3 days.

- if parasites are suspected, three specimens collected within 10 days should be

sufficient for microscopic detection of ova and parasites.

- The newer methods detect parasite antigens, and one sample is usually sufficient.

-commercial systems are available with preservatives for bacteria and parasites.

The appropriate ratio of stool to preservative is 1:3

Feces sample: for gastrointestinal tract infection we take either rectal swab or feces

sample.

Rectal swab: from the rectum. مسحات شرجية()

We should not use carton container for watery stool (diarrhea) because it contain

fiber that will absorb the water and leakage will occur. so we use plastic container .

Three states of the Stool:

Formed

Semiformed

Watery (diarrhea)

For formed or semiformed stool we can use the fiber container (carton) but for

watery stool we have to use a plastic container.

A single specimen or swab does not exclude the infection.

Parasite: microscopic exam is important.

Microscopic exam is not very important in bacteria sample but culturing is important

Microscopic exam culturing biochemical tests

Question by the Dr: if we are looking for bacteria the microscopic examination is not

necessary on the other hand if we are looking for parasite infection it is important?

In the intestine we have normal flora (one of them E.coli) how can we recognize if

this E.coli is toxigenic or cause food poisoning etc.. All of them are gram negative so

we can't recognize them in the smear, it does not give any indication, but parasite

are not normal flora and are recognizable.

There are other methods to detect parasite in stool depending on:

1) Serology: (antigen / antibody) reaction.

2) Molecular polymerize reaction.

Sometime we need to put the stool (or urine) in preservative media but other

samples do not need preservation.

The ratio of Bolic acid: for urine or stool sample preservation is 1:3 .

Fungal scrapings

- Wipe nails or skin with alcohol , use clean, screw-cap container.

A) hair/nails/skin hair: 10-12 hairs with shaft intact

b) nails: clip affected area

c) skin: scrape skin at outer edge of lesion

Hair should be extracted with it's follicle (the area would have pus , very soft and the

hair is easily removed) .

Genitalia

A. Cervix/vagina: Remove mucus before collection; do not use lubricant on

speculum; swab endocervical canal or vaginal mucosa, swab transport system or

JEMBEC transport system

Genitals: most samples are HVS (high vaginal swab).

Vagina/cervix sample: taken by the gynecologist not by the technician by using

speculum that is inserted inside the vaginal canal then the OS ( opening of the cervix)

will appear then we clean the area with cotton swab to remove all excretion and

debris then we insert another clean swab inside the OS .

If there is a case of gonorrhea we should use JEMBEC transport media (plate special

for isolation of pathogenic Neisseria which causes gonorrhea).

JEMBEC transport: rectangular in shape containing chocolate agar and special tablet

(CO2 generating tablet) which is needed by the capnophilic bacteria ( 5%-10%) co2 .

B. Urethra : Flexible swab inserted 2-4 cm into urethra for 2-3 sec or collect

discharge, swab transport system or JEMBEC transport system

Lesion/wound/abscess Wipe area with sterile saline or alcohol , superficial swab

along outer edge, swab transport system, deep aspirate with needle and syringe,

anaerobic transport system

urethra : male sample , if the male suffer from gonorrhea there is certain swab ( not

the normal swab that is used) consisting of a fine wire containing a small swab which

is inserted inside the opening of the urethra , sometime there is extra discharge

produced extracellular from the male genital tract and sometimes there is none so

we can insert the swab inside the urethra , get the sample and directly striking on

jembec transport system "chocolate agar" .

For anaerobic infection: a deep aspirate with syringe because we have mixed

infection and there is too much pus.

jembec agar for Neisseria ( appear small shiny colonies) provide two factors that

affect the growth of Neisseria : 1)X factor 2)V factor . "We will take about them

later on"

Respiratory: 1) upper respiratory tract infection (sore throat, tonsillitis, pharyngitis)

2) Lower respiratory tract infection (pneumonia, bronchitis)

Lower bronchial specimens Respiratory tract:

- sputum, rinse mouth or gargle with water, instruct to cough deeply into container.

Or the patient should rinse the mouth with water and expectorate with the aid of

a deep cough directly into a sterile container (expectorated sputum).

- Patients with dentures should remove the dentures first. A single specimen should

be adequate for detection of bacterial LRT infection. If fungal or mycobacterial

infections are possible, three separate early morning specimens (collected on

successive days) are appropriate.

- Specimens may be collected through aerosol-induction in which the patient

breathes aerosolized droplets of a solution that stimulates cough reflex (induced

sputum).

If we are talking about lower respiratory tract specimen: then we are talking about

sputum which is different form the saliva. (It's difficult specimen to take, if there is

any contamination of normal flora from the upper respiratory tract then the sample

is rejected) the sample should be obtained from deep.

Pneumonia in the case of tuberculosis the media used is Lowenstein – Jensen media

where the patient can spit on.

Pneumonia ( streptococcus pneumonia , haemophilus influenzae ..) : a single

specimen should be adequate ( from the lower respiratory tract ).

Fungal infection such as histoplasmosis capsulatum , Cryptococcus neoformans and

mycobacteria tuberculosis three sample on three successive days ..

NAOH: is inhaled which cause irritation in the respiratory tract and cause cough

reflex "induced-sputum"

Respiratory tract: uRT

1. Nasal : Insert premoistened swab with sterile saline 1 inch into nares, swab , use

transport system

2. Nasopharynx : Insert flexible swab through nose into posterior nasopharynx,

rotate for 5 sec, swab transport system or direct inoculation to media

3. Throat Swab, Posterior pharynx, tonsils, and inflamed areas, swab transport

system

Disinfect skin; do not allow tissue to dry out; if necessary, moisten with Tissue:

sterile saline, use transport system or sterile screw-cap container

.

We take the sample from the area which appears inflamed where redness, pus and

ulcers are observed.

If there is tissue ( not swab ) inflammation scraps are taken and put in a container

with normal saline to keep the tissue moist then transport it to the lab and do

homogenization and divide it to small pieces then we use centrifusion and take the

superlative and culture it on agar .

Lumber puncture : taken by the physician between L3-L4 or L4-L5 , this is the most

dangerous and valuable sample so we have to be sure there is no contamination at

all ,Three samples should be taken:

1)for biochemistry tests

2) Hematology

3) Microbiology

And that's because Not only the presence of microorganism is important but the

levels of glucose and protein in the CSF is important too.

Urine SAMPLES

1. Clean-catch midstream: Clean external genitalia; begin voiding and after several

ml have passed; collect midstream without stopping flow of urine, sterile, screw-

cap container, collect 2-3 ml. The first portion of the urine flow washes

contaminants from the urethra, and the midstream portion is more representative

of that in the bladder.

Urine: the most famous, widely used sample is the clean – catch midstream sample

(early morning midstream) because in the early morning the levels of microorganisms

are at their highest and the patient drink solution and water during the day so the

levels are lowered after the morning.

The first 1/3 of the urine is excluded because it contains the normal flora in the

urethra and first part of the bladder so we take the second 1/3 which contains the

pathogens microorganism.

The urine is collected in special screw-cup container with graduation.

We do both micro and macroscopic exam (macro: the color, turbidity, glucose and

protein level using strips) then culturing.

The contamination level is 24% .

2. Catheter Clean urethral area, Insert catheter, and allow first 15 ml to pass; then

collect remainder sterile, use screw-cap container or urine transport kit

Catheter clean urethral area: for people with bed rest or had gone under surgery and

can’t go to the bathroom.

Catheter: from urethra opening to plastic bag for collection of the urine and at the

end of the day the bag is removed.

This method usually contains a lot of contamination.

3. Indwelling catheter, Disinfect catheter collection port, aspirate 5-10 ml with

needle and syringe, use screw-cap container or urine transport kit

Indwelling catheter: catheter is fixed in the bladder we draw the sample using syringe

aspirate (5- 10 ml)

4. Suprapubic aspirate, Disinfect skin, aspirate with needle and syringe through

abdominal wall into full bladder, use sterile, screw-cap container or anaerobic

transport system

Superpubic aspirate: from the bladder ( upper genetalia ) taken by the physician by

syringe . (Need to drink lots of water)

Mostly used in babies who is under two years old with urinary tract infection.

The level of contamination is the lowest between all methods (1%).because it is

taken directly from the bladder and does not pass throw urethra.

Patient specimens or culture isolates must be TRIPLE PACKAGED BEFORE BEING

SHIPPED.

The material is placed into a Primary receptacle that must be watertight. Absorbent

material is placed around the primary receptacle, and it is then placed into a

Secondary container that is also watertight. The secondary package is sealed and

placed into a sturdy Outer container constructed of fiberboard.

hazardous Specific instructions must be followed for labeling the container as “

.”material

Specimen transport: when the specimen is taken in the clinic and need

transportation to the lab.

The sample should be kept in 3 containers and label the last container the hazardous

material considering that any sample is infectious.

When the sample reaches the lab how the technician deals with it: four levels

Specimen Priority

A four-level scheme of prioritization may be used based on the critical

nature of the specimen or potential for specimen degradation.

Critical/invasive ( amniotic fluid, blood, CSF, heart valves, Level 1:

pericardial fluid

should deal with it immediately

fluids, bone, drainage from wounds, feces, sputum, Unpreserved ( body Level 2:

tissue) feces not preserved .

): Quantitation required (catheter tip, urine, tissue for quantitation Level 3

: Preserved ( feces in preservative, urine in preservative, swabs in holding Level 4

medium, (aerobic and anaerobic) need transport media .

Otherwise the sample is put in the refrigerator not the freezer and for 24 hour

maximum.

Macroscopic Observation

Notations from the macroscopic observation should include the following:

• stool consistency (formed or liquid) also the smell ( strong smell of stool is an

indication of giardia infection caused by parasites ) and color .

• blood or mucus present

• volume of specimen : important for semen sample ( should be a specific volume

otherwise there is abnormality ).

• fluid: clear or cloudy "if the urine is cloudy this indicate that there is infection "

areas of blood and mucus are selected for culture and direct microscopic

examination.

Anaerobic cultures may be indicated if gas, foul smell, or sulfur granules are

present.

The diagnosis is evident if adult helminths or tapeworm proglottids are present in

the specimen.

Progolottids : parasitic infection . ية :ديدان شريط

Microscopic Observation

A direct microscopic examination is a useful tool that provides rapid informations

(1) it can be used to determine the quality of the specimen. Sputum specimens that

represent saliva rather than lower respiratory secretions can be determined by the

quantitation of WBCs or epithelial cells.

(2) it can give the indication of the infectious process involved. Gram stain of a

sputum specimen revealing WBCs and Gram-positive diplococci is indicative of

streptococcus pneumoniae.

(3) the routine culture workup can be guided by the results of the smear.

(4) it can dictate the need for nonroutine or additional testing. The presence of

fungal elements in a specimen for bacterial culture will alert the technologist to

notify the physician to request a fungus culture.

Sometime we have fastidious bacteria it’s not necessary to use fastidious plate we

use plates for all purposes that provide the lowest nutritional needs but using gram

stain we can know if we have fastidious sample from the shape of the bacteria such

as haemophilus influenzae rod , filament .. so we use chocolate agar or agar that

contain NAD(factor V) and hemin (factor X) .

Fungal media is completely different from bacteria media and is recognizable

because the size is larger and the shape (filaments, hypha ...) from the gram stains.

Culture Media Selection

- The selection of media to inoculate is based on the type of specimen submitted

and the organisms likely to be involved in the infectious process.

- Specimens in which fastidious pathogens are more likely involved require media

with appropriate nutrients to aid in their recovery.

- Specimens that are collected from a site containing normal biota will require

types of media to diminish the normal biota while allowing the pathogens to be

detected.

Such as in case of salmonella we use bismuth sulfite agar which inhibit the growth of

normal flora and allow only salmonella to grow.

The routine primary plating media include

• Nonselective agar plate ( for all organism)

• Enriched medium for fastidious organisms

• Selective and differential medium for enteric gram negative bacilli for most

routine bacterial cultures ( MacConkey agar)

Selective medium for gram-positive organisms for specimens in which mixed gram-

positive and gram negative bacteria are found.