1. Microbiology Lesson 2: Specimen Collection and Testing Procedures45
Microbiology
Lesson 2:
Specimen Collection and Testing Procedures

2. Lesson Objectives
Upon completion of this lesson, students should be able to …
Define and spell the terms to learn for this chapter.
List general guidelines for obtaining specimens.
Describe the different growth media needed for culturing microorganisms.

3. Lesson Objectives Explain the purpose of obtaining a specimen.
Understand how cultures are interpreted.
Define sensitivity testing and explain how it is done.
Explain the importance of the Gram stain.
Describe the basis for serological testing and name three examples performed in a POL.

4. Specimen Collection and Transportation
No shortcuts
Steps:
Educate patient properly
Follow guidelines carefully
Complete label and/or requisition carefully
Transportation
Maintain appropriate environment and temperature for survival

5. Critical Thinking Question
What could be the result of an improperly educated patient?

6. Information Required on a Specimen Label
Patient’s full name
Time of collection
Type of specimen
Antibiotic treatment in use, if any
Your initials

7. Information for the Laboratory Requisition
Patient’s address
Identification number
Age and gender
Insurance information
Type and source of the specimen
Test requested
Medication patient is currently receiving
Diagnosis, if available
Physician’s information (name, address, telephone number)
Special information or orders

8. Collection Devices Culturette system: Sterile containers
Sterile swab
Sealed plastic vial of medium
Used for many types of specimen collection
Sterile containers
Large sterile containers

9. Collection Devices

10. Diagnosing Infection Patient is examined
If necessary, specimens collected and prepared for transportation
Culture may be necessary–incubated for 24 hours
Proper antibiotic determined
In some cases a wet mount is used

11. Microbiology Equipment and Procedures
Inoculating Equipment:
Loop
Needle
Incineration equipment
Culture Media:
Solid (slant)
Semisolid (agar)
Liquid (broth)

12. Critical Thinking Question
How should you decide which type of culture media to use?

13. Common Culture Media Blood agar Chocolate agar EMB MacConkey agar
Thioglycollage broth
GN broth

14. Microbiology Equipment and Procedures
Inoculating Media:
Inoculating loop used to create streak culture
After inoculation, Petri dish is placed in incubator to allow organism to grow

15. Microbiology Equipment and Procedures
Sensitivity Testing
Determines which antibiotics will kill bacteria
Tools:
Petri dish
Mueller-Hinton agar
Direct Examination
Direct smear
Wet mount preparation

16. Direct Smear Process Perform hand hygiene and apply gloves
Assemble equipment
Label clean slide
Inoculate slide
Allow slide to air dry for 20 to 30 minutes
Hold slide with thumb forceps and pass over Bunsen burner flame
Slide is ready to be stained

17. Wet Mount Slide Preparation Process
Perform hand hygiene and apply gloves
Label dry slide
Inoculate dry slide
Place drop of saline solution on top of specimen
Place cover slip on to smeared slide
Physician or lab specialist will then examine slide

18. Gram Stain
Differentiate, or separate, bacteria into two groups: gram positive and gram negative
Different bacteria stain differently, depending on the compounds in their walls
Gram-positive bacteria retain the crystal violet-blue color and gram-negative bacteria retain only the pink safranin color
Thus, gram-positive (violet) bacteria can be distinguished from gram-negative (pink) bacteria
Gram stains must always be accompanied by culture for microorganism identification

19. Staining Specimens

20. Types of Specimens
Throat
Sputum
Urine
Stool
Wound

21. Obtaining a Throat Culture

22. Throat Specimens
One of the most frequently requested specimens in a POL
Based on signs and symptoms the patient presents with the physician will order to identify the pathogen involved and begin treatment.
Confirmation of Streptococcus pyogenes is important because of its virulence and possible complications
When performing a throat culture, it is important not to touch the insides of the mouth or the tongue with the swab to avoid contaminating it

23. Obtaining a Sputum Specimen

24. Obtaining a Sputum Specimen
The mucous substance expelled by coughing or clearing the bronchi
To obtain it the patient must be carefully instructed to cough deeply and spit up the coughed up material into a sterile container
Explain to the patient that this should not be saliva from the mouth

25. Obtaining a Sputum Specimen
Often it is possible to obtain a good sputum specimen if the patient is reminded to try to collect it on rising in the morning
The purpose for obtaining a sputum specimen is to isolate and diagnose diseases such as streptococcal pneumonia, influenza, and tuberculosis

26. Urine Specimen
For culture it must be either a catheterized specimen or a clean-catch midstream sample (CCMS)
Both methods provide sterile samples
Any other type of urine specimen (one for routine analysis for example) would be contaminated by organisms in the container or on the hands or genitals of the patient

27. Urine Specimen
In doctors’ offices and smaller facilities, self-contained culture units are purchased and used
Often urine cultures require a means to provide a quantitative result of the number of microorganisms in the sample

28. Collecting a Stool Specimen

29. Stool Specimen
May be tested for bacterial, parasitic, or protozoal infections, for the presence of occult blood, and for excessive amounts of fat steatorrhea
Collection varies with the type of test ordered
Correct collection is critical to an accurate result
Fecal specimens must be free of urine or water from the toilet, and toilet tissue

30. Occult Blood
A stool specimen is required to test for occult or hidden blood that may indicate bleeding in the gastrointestinal tract
Often the patient is given the test units to take home and collect the specimen
Directions are provided on each test unit, However, instructions should be reviewed each time they are given to a patient
Patients are instructed to write their name, date, and doctor’s name on label of the unit

31. Occult Blood
Using one of the wooden spatulas provided, they are to collect a small amount of stool and place it in one of the circles on the back of the booklet
Another sample from a different area of the stool should be obtained, and placed on the other circle
Patients should close the unit and take it or mail it to the doctor’s office or the laboratory as requested

32. Occult Blood
For more accurate results patients should be instructed to refrain from vitamin C and red meat for 3 days prior to testing because they may cause false positives
It is important to check the expiration date of any test kit before giving it to the patient

33. Stool Specimen for Ova and Parasites
The presence of microbial organisms, such as ova and parasites (O&P), may be determined by testing feces or stool
The presence of ova or eggs or other forms of a parasite indicate parasitic infestation
Identification of the parasite aids in selecting the correct treatment

34. Stool Specimen for Ova and Parasites
Commercial kits are available that provide containers for fresh stool specimen and two additional vials for preserved specimens, one containing formalin and the other containing polyvinyl alcohol

35. Swabs for Obtaining Wound Specimens

36. Wound Specimens
Sterile swabs are used to obtain a specimen from a wound, abscess, or incision to test for pathogenic microorganisms
The procedure is similar to obtaining a throat culture
Several specimens may be necessary from different locations
Be certain to label each appropriately as to the source

37. Collection of Cerebrospinal Fluid (CSF)
Always treated as a stat procedure
Procedure is uncomfortable for the patient, and the specimen must be handled with care
Usually three tubes are collected under sterile conditions and sent for testing
The culture and sensitivity test should be performed before chemical and other tests using the second of the three tubes
Tubes one and three are more likely to be contaminated because of the entry and removal processes of collection

38. Strep Test

39. Group A Strep Screen Test that is done frequently in POLs
Especially efficient in the pediatric office because it is self-contained and can be done while the patient waits
Screen is an antigen detection test for group A beta-hemolytic Streptococci and follows the general procedure for antigen-antibody agglutination (clumping together) tests, which produce a clumping of cells

40. Group A Strep Screen
There are many CLIA waived group A strep kits available that test for the extracted Group A beta-hemolytic Streptococcus antigen

41. Questions? 41