Clinical Specimens

Clinical Specimens

Background Information

In today’s laboratory exercise we will be examining several common specimens routinely found in the clinical laboratory. While it would be impossible in one lab period to discuss and examine all the various possible types of pathogens associated with these specimens, we will take the opportunity to look a few of the most common infectious agents associated with them and some of the basic techniques in collecting and running up the specimens.

Infections of the upper respiratory tract are very common. Viruses cause many of these infections, but bacteria are responsible for a large number as well.

Proper diagnosis of upper respiratory tract infections is important so that the appropriate treatment can be administered. One of the best ways of isolating bacteria from a patient is to perform a throat swab. Bacteria picked up on the swab are placed onto a panel of media and incubated. The media are then examined for potential pathogens. All suspected pathogens are identified and their sensitivity to antibiotics is determined.

Urinary tract infections are extremely common and as such many urine specimens will enter the clinical microbiology laboratory. Most of these infections are caused by a handful of organisms which include E. coli, Proteus sp., N. gonorrhoeae, groupB Streptococcussp. and enterococci.

While these are common isolates, they by no means constitute the only organisms found in urine. It should also

be noted that simply finding bacteria in urine is not always an indication of infection. While it is too complicated to explain all the parameters and considerations in interpreting urine specimens at this point there are two basic characteristics that tend to indicate an urinary tract infection; > 103uropathogens (pathogens that normally cause urinary tract infections) and the presence of white blood cells.

The presence of bacteria in stools is to be expected and usually is not a significant finding unless the bacteria are known pathogens. Stool specimens may also contain parasites, or parasite ova, fungi, or viruses. Since, there is a wide range of potential pathogens in stool; a specimen may need to be worked up to check for bacteria, fungi, parasites and viruses. In this laboratoryexercise we will focus only isolating bacteria from the stool. It should be noted that the stool should be worked up as soon as possible.This reduces the chances that pathogen will die off and/or overgrown by normal flora.

For safety sake the stool and urine specimens used in today’s laboratory exercise are not real, but they do contain pathogens. The throat specimens and nasal specimens are real and may contain dangerous pathogens and must be handled with great care.

Please be careful with them.

Purpose

The purpose of this laboratory exercise is to instruct the student in the proper method of collecting specimens from the throat and nasal passage. We will attempt to identify some of the normal flora organisms associated with these specimens.

We will also be examining urine and stool specimens for pathogenic bacteria.

Methods and Materials

Materials

1)Blood agar plates

2)MSA plates

3)MAC agar plates

4)EMB plates

5)B-G plates

6)Bile-esculin agar plates

7)Optochin disks

8)Bacitracin disks

9)Sterile saline

10)Sterile swabs

11)Oxidase reagent

12)Microscope slides

13)Gram stain reagents

14)Inoculating loop

15)Bunsen burner

16)Hydrogen peroxide

17)Commercial identification kits

18)Sterile pipettes

19)Pipettor

20)Test tube rack

21)Parafilm

22)Toothpicks

23)Wooden tongue depressors

24)GasPak Chamber and CO2

Protocol - Day One Throat and Nasal Swabs

1)Using the tongue depressor to hold down your lab partner’s tongue, take the sterile swab and gentlyswab the back of their throat. Be careful not to probe too deeply, as this will elicit a gag response.

2)Immediately after swabbing the throat, roll the swab over the edge of the blood agar plate, contacting no more than a centimeter or so of the medium.

3)Using the inoculating loop, streak the plate for isolation.

4)Repeat steps 1 through 3 using a MSA plate.

5)Place the blood agar and MSA plates into the incubator.

6)To perform the nasal swab, carefully place a sterile swab into the nostril and gently swab.

7)Repeat steps 2 and 3 using blood agar and MSA.

8)Place the blood agar and MSA plates into the incubator.

9)Be sure to properly label each plate

Urine Specimens

1)Obtain a prepared urine specimen from the instructor.

2)Prepare Gram stains of the prepared urine specimen. Be sure to note the typical morphology of each organism and its staining characteristics. Record your results in Table 2.

3)Inoculate a sample of the prepared urine specimen onto the appropriate media: MAC, Blood agar, Bile-esculin agar. The instructor will demonstrate the proper inoculation techniques.

4)Incubate the plates at 37oCovernight.

Stool Specimens

1)Obtain a prepared stool specimen from the instructor.

2)Inoculate the enteric media: EMB agar MAC agar, and B-G agar, as directed by the instructor.

3)Incubate the plates at 37oCovernight.

Please note that you will not be using all the various types of media for all the specimens. Use only the appropriate media for the particular specimen.

Protocol – Day Two

1)Examine all the plates. Record your results in Table 1.

2)Select several interesting colony types and make an attempt to identify them. Perform Gram stains on the organisms and record your results in Table 2.

3)Set up and perform any necessary tests and record your results in Table 3.

NOTES:

Results

Table 1 – Colony Morphology and Characteristics

Medium / Throat / Nasal / Urine / Stool
Blood agar / N/A
MSA / N/A / N/A
Bile- esculin agar / N/A / N/A / N/A
MAC
agar / N/A / N/A
EMB
agar / N/A / N/A / N/A
B-G
agar / N/A / N/A / N/A

Table 2 – Gram Stains

Throat Culture / Nasal Culture / Urine Culture
Day One
Day Two

Table 3 – Tests Results

Throat Culture / Record the tests you performed and results
Nasal Culture / Record the tests you performed and results
Urine Culture / Record the tests you performed and results
Stool Culture / Record the tests you performed and results

Questions

1)What types of organisms would you normally expect to find the throat?

2)Did you find any of the expected organisms?If so, which ones?

3)Did you discover different types of organisms on the various media used?

4)What types of organisms would you normally expect to find in urine and feces?

5)Did you find any of the expected organisms?If so, which ones?

6)Did you discover different types of organisms on the various media used?

Specimencollectionandprocessingprotocol

The following protocols are general protocols used to acquire a variety of specimens. They are here to give you an idea of what goes into collecting clinical specimens and some of the types of organisms you might expect to find. More specific protocols may be needed under special circumstances.

Blood Cultures:

1.The usual method of preparing the arm with tincture of iodine should be followed.

2.An Isolator tube should be drawn. Disinfect the STOPPER with iodine and collect the sample through that end using the closed Vacutainer system or needle and syringe. It is important that the tube be thoroughly mixed before sending it to the laboratory.

3.At least one full tube of blood is required on all patients. Tubes containing less than 5 ml of blood cannot be processed. 1.5 ml pediatric tubes are available for patients less than 5 years old. Only blood or bone marrow should be put into the Isolator tubes.

4.Send the Isolator tube with a patient label attached, along with requisition, to the Clinical Microbiology Laboratory.

5.If the specimen is to be cultured for fungi, a second tube and explicit request for fungal culture is necessary to indicate to the laboratory personnel that the specimen is to be so processed.

Each blood specimen is routinely cultured both aerobically and anaerobically. Gram stains are not performed on primary blood samples submitted for cultures.

Blood cultures are examined twice daily and Gram stained if suspicious. Positive findings are immediately phoned to the house officer or unit charge nurse. If no growth appears, the final report is issued on the fourth day.

Bone Marrow:

Bone marrow samples are collected into a pediatric (1.5 ml) Isolator tube and processed as blood cultures.

Remember that bone marrow specimens are excellent specimens for acid fast and fungal cultures.

CSF/Body Fluids:

CSF, biopsies, and fluids from normally sterile body areas should be sent to the lab without delay in a sterile screw-capped tube. These are considered STAT and are processed immediately. Gram stains are routinely performed on all such specimens. These specimens are held for 72 hours before being reported as negative. Any growth on these specimen types is Gram stained and the physician or unit charge nurse notified immediately. Aerobic and anaerobic cultures are routinely performed on all such specimens.

It is known from past experience that the number of infecting organisms per ml of peritoneal fluid is usually quite low.

In order to provide optimum culture conditions, we request that at least 100 ml (or preferably the whole bag of fluid) be submitted to the laboratory for culture.

Exudates:

Swabs from exudates (throats, wounds, and genitals) are to be submitted in transport medium. Exudates from wounds should be submitted in special anaerobic transporters available in the laboratory.

1.Any specimen in which gonorrhea is suspected should be so noted on the requisition to insure proper laboratory processing.

2.Gram stain requests on cervical smears for gonorrhea cannot be accepted; cultures should be performed.

3.In order to speed the reporting of results of female genital cultures, the term "NormalVaginal Flora" will be employed whenever a mixture of two or more of the following organisms are recovered:

Staphylococcusepidermidis

LactobacillusStreptococcus viridans group

Acinetobacter

Proteus sp.EnterococciColiformsVeillonella

YeastsDiphtheroidsNeisseria sp.(other than GC and Meningococci)

Since these organisms do not usually cause disease of this organ system, antibiotic susceptibilities will not routinely be performed.

4.The male genital secretions are normally sterile; however, Staphylococcus epidermidis,diphtheroids, andStreptococcus viridansgroup may be found as normal skin flora. Sensitivitytests are not routinely doneon these. The usual pathogen sought isN.gonorrhoeae, although others may include species ofStreptococcus,S. aureus, gramnegative rods, yeasts, and trichomonas. Sensitivities are done on all pathogens except gonococci, trichomonas, and yeast. All gonococci are tested for the presence of beta lactamase.

Fecal:

Routine adult fecal specimens are screened for the presence ofSalmonella, Shigella, E. coli 0157:H7 and

Yersiniaonly. Other enteric pathogens such as Vibrio species,Clostridium difficile, S.aureus, Campylobacter, or yeast must be indicated on the requisition to facilitate properlaboratory processing. Routine gram stains are not done on stool specimens unless Staphylococcus, or yeast are suspected.

Sputum:

Expectorated sputum must be collected in the Falcon sputum collection system. Since sputum is not a normal secretion, the term "normal flora" is meaningless. However, as the sputum passes through the oropharynx, it becomes contaminated with the commensal bacteria of that region. Thus, sputum cultures growing out primarilyStreptococci of theviridans group,Neisseria sp., S. epidermidis, or diptheroids will be reported as "normalthroat flora".

Throat:

Normal throats, cultured on sheep blood agar and incubated aerobically, invariably grow out Streptococcusviridans and Neisseria sp. (other than meningococci and gonococci). Cultures of normal throats may also grow out some:

S. epidermidis

diphtheroids

S. aureus

coliforms

Hemophilus influenzae

Proteussp.

Hemophilus parainfluenzae

Candidasp.

pneumococci enterococci

nonhemolytic streptococci

Infected throats commonly grow out beta hemolytic Streptococci (usually group A) or a predominance ofS. aureus, pneumococci,H. influenzae, coliform orCorynebacteriumdiphtheriae.

Urine:

Urine specimens are not Gram stained unless specifically requested.

Submit the specimens to the laboratory in the sterile urine cups which are available from NCBH Storeroom.

Since the external urethra has a normal flora, it is important that the patient be instructed to properly cleanse the area before collecting the specimen. The specimen should be brought to the lab within one hour and processed as soon as possible to avoid overgrowth of these organisms.

The calibrated loop method is used for the semi quantitative cultivation of clean-voidedand catheter-obtained specimens.

Acute urinary tract infections are generally caused by a single organism in numbers exceeding 10,000/mL. On occasion, two organisms may cause an infection; more than two organisms causing infection is very unlikely.

Negative cultures are reported as no growth in 24 hours.

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