Chapter 42 Notes: Specimen Collection, Stool Tests, Cultures, Stains, TB, and PPD
Fecal Specimens: Collection, Handling, and Practicalities
Context: Chapter 42 content focusing on collecting fecal specimens; information used for Chapter 44 test.
Why fecal specimens are collected:
Check for occult blood (guaiac/hemoccult), ova and parasites (O&P), and bacterial or viral infections.
Specimen containers and preservatives:
Common practice: urine container can be used for some tests (e.g., C. diff) when sterility is not required.
Special containers with preservatives are used for O&P testing and stool cultures.
Top color indicates test type:
Orange and white tops: used for stool cultures.
Pink and gray tops: used for ova and parasite (O&P).
Preservatives in containers are test-dependent; follow the test requirements.
Home collection guidance:
Verify the patient is collecting the correct type of specimen and whether refrigeration is required.
Stool samples are usually not refrigerated unless the test protocol requires it.
Ensure proper collection amounts: some tests require small amounts, others require more material (e.g., bacterial/viral tests, O&P).
Do not urinate during stool collection; urine can destroy microorganisms and contaminate the specimen.
Lab personnel may look for discoloration or pus in urine, which would indicate contamination if urine were present.
Three consecutive stool specimens are typically collected, but with a specific rule:
Three consecutive days are not required to be consecutive calendar days.
Example: if a patient has stools on Monday, Wednesday, and Friday, collect specimens on those days (Monday, Wednesday, Friday).
Three-sample practical rule:
Only one specimen should be collected per day.
If there are three bowel movements in one day, you do not collect multiple samples from that single day.
Do not have to be three consecutive days of sampling; rather, the collection can be spaced out over a period of time as needed to ensure adequate representation of the patient's condition. This approach helps to minimize patient discomfort while still obtaining sufficient data for accurate analysis.
Guaiac (hemoccult) testing: hidden blood in stool
Purpose: A screening tool for colon cancer and other sources of GI bleeding. This non-invasive test is performed by applying a small sample of stool to a test card and following the manufacturer's instructions; a color change indicates the presence of blood.
Principle: Guaiac reagent turns blue when oxidized in the presence of blood.
Home/office procedure (hemoccult):
Collect a very small amount of stool using a disposable stick; place on the two windows of the testing card.
Turn the card over and apply the color developer on the other side (for developing the test).
A control area is included to verify test validity.
Reading results: if blue develops on the test window(s), there is evidence of blood in the stool.
Example results images described:
Negative: no blue color in the test window.
Positive: blue color appears in one or both test windows.
Practical application and interpretation:
A positive guaiac test may lead to further diagnostic workup, such as colonoscopy.
Pre-test patient education and dietary restrictions to reduce false positives/negatives:
Seven days before testing: avoid aspirin, iron supplements, and large doses of vitamin C (gastric bleeding may mask lesions).
Two days before testing: avoid red meats and processed meats.
Two days before testing: avoid turnips, broccoli, cauliflower, and melon.
Maintain a high-fiber diet leading up to testing.
Do not begin testing until three days after the menstrual cycle.
Drink plenty of fluids to prevent constipation.
Store slides at room temperature; protect from heat, sun, and fluorescent light.
Sputum specimens: indication and collection
Indication: Diagnostic analysis for unresolved cough with mucus production. Collection: Instruct the patient to cough deeply to obtain a good sample, ideally collecting the first morning sputum to ensure accuracy. Instruct patients to avoid eating or drinking for at least one hour prior to collection to enhance specimen purity.
Source considerations:
Upper respiratory tract samples are not sterile; lower respiratory tract samples are considered sterile.
Procedures for obtaining lower respiratory tract sputum:
Outpatient bronchial washing or brushing to obtain lower tract specimen.
Diagnostic scope for sputum:
Can detect cancers, viral infections, bacterial infections, fungal infections, and TB. Proper collection techniques are essential to ensure accurate results, including instructing the patient to cough deeply and expectorate into a sterile container.
Diagnoses are typically confirmed by a cytologist or pathologist.
Home collection guidance (when applicable):
Collect first morning sputum.
Rinse the mouth with water and spit out to minimize contaminating superficial cells.
Open sterile container without touching the inside of the container or lid.
Cough deeply from deep lungs into the cup; fill should not exceed half full.
Recap securely; include date and time of collection.
Store according to the test requirements.
Other lab specimens and cultures: overview
Pinworm testing:
usually infects small children but can affect individuals of any age, leading to symptoms such as anal itching and discomfort. Testing is done using tape tests or by examining stool samples, and treatments typically involve antiparasitic medications.
Pinworms are small, thin worms; diagnosis via identifying worms or eggs.
Life cycle: worms live in the intestines; eggs are laid in the perianal folds while sleeping.
Collection method: adhesive tape (scotch tape) around the anus on waking; press onto a slide and examine under microscope.
Alternative: test kit with a sticky paddle placed into a test tube to return to the lab.
Cultures:
Cultures are collected from sites that appear infected (throat, mouth, ear, eyes, nose, vagina, anus, wounds). Performed by qualified individuals.
Specimens collected in a culturette container; the end is popped to keep the specimen moist until lab processing.
Rapid group B (A) strep testing uses a sterile swab (not a cotton-tipped applicator) and yields results in minutes.
Throat swabs are collected for sore throat, fever, swollen glands, and cough. Rapid Goup A strep test. The actual organism that causes strep throat is group A beta hemolytic streptococcus bacteria, and we get it using a swab and you will swab the periconsular area in the back of the oral cavity.
And it's just the sides and do not touch with that swab, the lips, the cheeks, the gums, the teeth, or the tongue, because then you get normal flora and that will be tested, and we don't want to do that. After obtaining the specimen, it is crucial to properly transport it to the laboratory to ensure optimal viability for accurate testing. Additionally, if the initial test is negative but clinical suspicion remains high, it may be necessary to follow up with a throat culture for definitive diagnosis.
In clinical practice, rapid strep tests are often used instead of sending swabs to a lab for culture.
Nasopharyngeal swabs:
Collected with a thin, wired swab.
Used for several COVID-19 tests and other respiratory pathogens.
Blood cultures:
Blood drawn directly into a specially formulated broth in a vacuum bottle.
Culture and sensitivity (C&S):
Physicians order C&S to identify effective antibiotics for a detected organism, especially if initial antibiotics are ineffective.
Procedure sequence:
Collect specimen and inoculate onto culture media or agar.
For many pathogens, first culture is attempted; if nothing grows in ~48 hours, a sensitivity test may be performed.
Agar and streaking basics:
Used to see which anitibiotics are effective against the isolated organisms, guiding treatment options. It is crucial to follow proper techniques for obtaining specimens and handling cultures to prevent contamination and ensure accurate results.
Agar is a growth-supporting medium (solid or liquid) used to culture microorganisms.
Primary media support growth of many organisms; selective media promote some while inhibiting others.
In-office culture capabilities have diminished; specimens are often sent to labs for culture and sensitivity.
Inoculation technique described: ensure correct swab type, collect appropriate specimen, label agar, streak onto one-fourth of the plate, then incubate.
Incubation conditions: typically at around 98.6^ ext{^\circ F} (human body temperature) for growth.
Antibiotic discs on plates (e.g., AMOX, ZITH) are used to determine antibiotic susceptibility by observing zones of inhibition.
Interpretation basics:
If growth occurs up to the antibiotic disc: resistant
If growth is inhibited around the disc: sensitive
After incubation, plates are read, and the provider selects the antibiotic likely to be effective.
Culture media types and labeling:
Media can be in Petri dishes (red agar in many cases), tubes with broth, etc.
Proper labeling and biosafety handling are essential; specimens go to the lab in appropriate biohazard containers.
Gram staining and bacterial morphology (historical and practical context)
Gram staining: a differential stain used to classify bacteria as Gram-positive or Gram-negative.
Historical note: Graham staining (often called Gram stain) technique described by Hans Christian Gram in 1884 for identifying organisms such as pneumonia-causing bacteria.
Process overview (not performed routinely in some modern office settings):
Prepare a fixed smear on a slide.
Apply crystal violet (primary stain) for ~30 seconds; rinse.
Apply iodine as a mordant; rinse.
Decolorize with acetone or alcohol; rinse.
Counterstain with safranin for ~30 seconds; rinse.
Result interpretation:
Gram-positive organisms appear dark blue or violet.
Gram-negative organisms appear pink or red.
Morphology descriptions:
Cocci: round bacteria; shapes can be in specific arrangements such as diplococci, staphylococci (grape-like clusters), or streptococci (chains).
Bacilli (vaxilla): rod-shaped bacteria.
Spirilla: spiral-shaped bacteria (corkscrew appearance).
TB-specific staining:
Mycobacterium tuberculosis has a lipid-rich cell wall that is not well stained by Gram stain.
An acid-fast stain (e.g., Ziehl-Neelsen or auramine-rhodamine) is used for TB because acid-fast organisms retain the stain after decolorization.
TB testing and interpretation
TB organisms require acid-fast staining due to cell wall lipid content.
The main TB pathogen is Mycobacterium tuberculosis.
PPD (Purified Protein Derivative) skin test, Mantoux method:
Administration: intradermal injection of 0.1 ext{ mL} of PPD at the injection site.
Reading: induration measured in millimeters after 48–72 hours.
Positive threshold: induration greater than 5 ext{ mm} is considered positive in certain risk groups; smaller indurations may be considered negative depending on guidelines and risk factors.
Practical clinical context for PPD:
Used for TB screening and to identify individuals who may require further evaluation (e.g., interferon-gamma release assays, chest X-ray, or preventive therapy depending on risk assessment).
Summary of key numerical values and practical rules
Incubation temperature for culture work: 98.6^ ext{\circ}F
PPD intradermal test volume: 0.1\,\text{mL}
TB induration threshold: >5\,\text{mm} (positive in many risk groups)
Hemoccult reaction: blue color indicates blood in stool on test windows
At-home guaiac test development: color developer used on opposite side of test windows; one window may serve as a control
Stool sample timing rule: collect on three separate days; one specimen per day; if multiple bowel movements occur on one day, do not collect more than one sample that day
Practical implications for students and clinicians
Proper instruction for patients is essential to obtain usable specimens and avoid contamination (e.g., urine avoidance in stool tests).
Understanding test top colors and preservatives helps ensure correct test processing and sample integrity.
Pre-test restrictions for guaiac testing are important to minimize false positives/negatives due to dietary or medication factors.
Grasping culture and sensitivity concepts is critical for selecting effective antibiotics and interpreting results (zones of inhibition indicate susceptibility).
Recognizing the limitations of older laboratory techniques (e.g., Gram staining in some settings) helps contextualize current protocols and the shift toward molecular methods for some pathogens.
Connections to foundational principles and real-world relevance
Specimen collection quality directly affects diagnostic accuracy and patient outcomes.
The concept of targeted testing (cultures, O&P, guaiac) reflects tailoring diagnostics to suspected pathogens and patient history (e.g., colorectal cancer risk, travel, exposure).
Antibiotic stewardship hinges on C&S to guide therapy and reduce resistance development.
TB screening (PPD) remains a cornerstone of public health, requiring standardization of interpretation and follow-up.
Ethical and practical considerations
Patient confidentiality and safe handling of infectious specimens.
Informed consent and education about the purpose of tests and potential risks of collection or exposure.
Balancing test sensitivity/specificity with patient burden (e.g., multiple specimens, timing constraints).
Koh testing- KOH: used on vaginal spec. to test for fungi and spores. This method helps in diagnosing infections such as candidiasis, allowing for appropriate treatment interventions. KOH, clears debris like artifacts, hair, etc so you can see the yeast.
Wet mount: examining for microorganisms under the microscope. suspends the organism in a liquid to study. Typically, saline or water is used as the liquid medium, which helps maintain the organism's natural state for better observation. Additionally, wet mounts can also be used for motility tests, as they allow for visualization of the movement of motile organisms such as flagellates.