Microbiology Lab Exam Study Guide by Susan B

Tetanus Immunity Under what circumstances would you give a patient tetanus toxin? Never!!! It would kill your patient.

Tetanus Immunity Under what circumstances would you give a patient equine tetanus antitoxin? Only in emergency situations and only if the patient has had no previous exposure. It can lead to serum sickness, a type III hypersensitivity reaction with fever, arthritis, and acute glomerulonephritis due to the deposition of immune complexes.

Tetanus Immunity Under what circumstances would you give a patient tetanus toxoid? Give to patients with recent exposure to tetanus to induce antibody formation. It should always be given in emergencies along with the antitoxin, injected at a separate site on the body. Toxoid will act within days if the patient has no memory cells and within 3-5 days if previously exposed.

Tetanus Immunity Under what circumstances would you give a patient tetanus antitoxin (human)? Always give it when in doubt. It will transiently protect the patient from the fast acting toxin but will not induce active immunity.

Koch’s Postulates What is the basic premise behind Koch’s postulates? You need to be able to isolate a pure culture from an infected host if you are planning on identifying/studying the organism.

Oil Immersion Microscopy What level of magnification can be achieved with an oil immersion lens? X1000, which gives it the necessary magnification to view Gram stains of bacteria.

Gram Staining What is the method of gram staining? Methanol -> Fixes organisms on slide Crystal Violet -> Stains purple Iodine -> Fixes the crystal violet on the peptidoglycan layer Alcohol/acetone -> decolorizes Gram neg bacteria Safranin -> Counterstains gram neg bacteria red

Gram Staining What are the requirements of gram staining? The cultures must be young (12-18 hours) and spread thin to avoid problems decolorizing

Blood Agar Hemolysis How Does Beta Hemolysis appear? Give examples. Clear zone around colony due to total lysis of RBCs GAS (S. Pyogenes), GBS (S. Agalactiae), Staphylococcus Aureus, Listeria Mono.

Blood Agar Hemolysis How Does Alpha Hemolysis appear? Give examples. Green zone around colony due to incomplete lysis of RBCs Streptococcus pneumoniae, Viridans group Streptococcus

Blood Agar Hemolysis How Does Gamma Hemolysis appear? Give examples. No zone around colony due to no lysis of RBCs GDS (Enterococcus, Streptococcus Bovis)

Catalase Test How does the catalase test work and what does it tell you? Hydrogen Peroxide is added to a smear in order to detect the enzyme catalase. This test is used to differentiate between Gram (+) cocci. If bubbles appear, then the test is positive and the gram (+) bacteria must be staphylococcus.

Acid-Fast Staining How is acid-fast staining accomplished? What does acid-fast staining indicate? Method: Carbol fuscin/magenta -> primary dye stains pink Alcohol/acid -> washes the dye off everything but AFB Methylene blue -> counterstains everything else Indicates an acid-fast bacilli like mycobacteria tuberculosis

Coagulase Testing What does coagulase indicate? No zone around colony due to no lysis of RBCs GDS (Enterococcus, Streptococcus Bovis)

Bacteria Gram Positive Rods? Listeria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Nocardia

Bacteria Gram Positive Cocci? Staphylococcus Aureus, Coagulase negative Staphylococcus, Group A & B Streptococcus, Streptococcal pneumoniae, Entercoccus

Bacteria Gram Negative Cocci? Neisseria

Bacteria Gram Negative Rods? E. Coli, Klebsiella, Pseudomonas, Proteus, Salmonella, Shigella, Citrobacter, Burkholderia, Yersinia, Enterobacter, Haemophilus, Bordetella, Brucella, Francisella, Citrobacter

Strep Throat Why is it important to correctly identify strep throat? 15% of Strep Throat is caused by Streptococcus Pyogenes and if left untreated, may lead to sequelae such as acute glomerulonephritis and acute rheumatic fever (0.5 -3%)

Strep Throat How is strep throat classically diagnosed? Swab Pharyngeal tonsils -> Streak on agar -> incubate for a day. GAS (S. Pyogenes) will show beta- hemolysis. Gram + cocci in chains on gram stain, and reaction with antiserum for group A carbohydrate.

Strep Throat How does the Rapid Strep Test work? It is more specific but less sensitive. Test directly for group A antigen. Swab tonsils -> put on kit and read….

Specimen Collection What constitutes a good specimen? Collected with metal (scalpel, syringe) and has whole fluids and tissues.

Specimen Collection What constitutes a bad specimen? Collected with swabs, not whole tissues

Male Urethritis What are the symptoms? Purulent urethral discharge, dysuria, urethral pruritis and/or rectal or pharyngeal inflammation…

Male Urethritis What should be included in your differential diagnosis? PMN with gram (-) diplococci -> N. Gonorrheae PMN without gram (-) diplococci -> Chlamydia trachtomatis (until proven otherwise)

Male Urethritis What is the most common STD among men and women? Chlamydia. It is the most common cause of non-gonoccal urethritis in men

Male Urethritis What is the treatment for male and female N. Gonorrhea infection? Gonorrhea -> 3GC or cipro Chlamydia -> tetracycline or erythromycin Usually, you treat both (assume they come together), inform partners and contact health department (yeah, right)

Female Cervicitis What are the symptoms of cervicitis? What are the risks of having cervicitis? Inflammation with purulent discharge. Both gonorrhea and chlamydia are risk factors for PID which can lead to ectopic pregnancies, and infertility

Female Cervicitis How is cervicitis diagnosed in women? Culture for Neisseria Gonorrhea using selective media and/or tissue cell culture for chlamydia. *A gram stain of cervical exudate will not be helpful in this case because of the normal vaginal flora present.

Female Vaginitis Three major causes of vaginitis? Bacteria, protozoa, or fungus

Female STDs White cottage cheese-like discharge, pH=4.5. Using a KOH prep, you see budding yeast cells w/ pseudohyphae. What is your diagnosis? Candida Albicans. Treat patient with antifungals

Female STDs Frothy, yellow-green discharge with pH>4.5 Trichomonas vaginalis. Treat patient with metronidazole Note: Use wet prep for trichomonas

Female STDs Thin, white-gray, homogenous discharge with pH>4.5 and visible “clue” cells Bacterial vaginosis. Treat patient with metronidazole or clindamycin

Female STDs Thin, white-gray, homogenous discharge with pH>4.5 and visible “clue” cells Bacterial vaginosis. Treat patient with metronidazole or clindamycin

Tuberculosis Most common presentation? Prolonged cough, chest pain, hemoptysis, cachexia

Tuberculosis How to diagnosis TB? NAAT, CXR, skin test, sputum culture, acid fast sputum stain (3 consecutive morning collections)

Tuberculosis What are the limitations of the NAAT test? 95% sensitive when given a AFB positive sputum sample and a culture will still have to be done in addition to this for specific identification of the agent.

Legionella pneumophila What symptoms are associated with Legionnaire’s disease Sudden onset cough, fever, chest pain, Chest X ray shows consolidation

Legionella pneumophila How can you diagnosis Legionnaire’s disease? Urinary Ag test (sensitive and specific), since legionella doesn’t stain well, and flourescent Abs are not very sensitive.

Laboratory Testing What causes most lab errors? 50% of lab errors are pre-analytical.

Laboratory Testing What can reduce the amount of lab errors? Label the specific source, pt immuno status, special requests for tests

GI Flora What do neonates have in their intestinal flora? Nothing! It’s sterile

GI Flora How do neonates acquire their GI Flora? From food, environment, and the birth canal (gross…)

GI Flora Where in the GI tract are most of the normal flora found? Lots of flora in lower GI, only a few in upper GI

GI Flora What type of flora predominate the GI tract? Anaerobes They outnumber aerobes x1000

GI Flora What type of pathological finding are anaerobes primarily responsible? Intra-abdominal abscesses, peritonitis, C. diff pseudo colitis

GI Flora What type of pathological finding are aerobes primarily responsible? UTIs and sepsis

GI Flora buzzwords… Diarrhea, chicken…………… Bloody diarrhea………………. Antibiotics, diarrhea………… Diarrhea, shellfish, DC……. Hemolytic uremic syndrome Appendicitis, diarrhea………. Salmonella, campy EHEC, STEC C. Diff Vibrio Shigella, EHEC/STEC Yersinia enterocolitica

Differential Plate Diagnosis What are HEA plates useful for recognizing in stool cultures? Both Salmonella and Shigella Only some gram (-) can grow on HEA

Differential Plate Diagnosis How do Shigella and Salmonella appear on HEA? Both Salmonella and Shigella do NOT ferment lactose -> grow green on HEA plate -Salmonella produces H2S -> black spot on green growth

Differential Plate Diagnosis Is HEA selective or differential? Both!!!!!

Differential Plate Diagnosis Is Campy Blood Agar selective or differential? It is selective for campylobacter

Differential Plate Diagnosis What agar is used to differentiate EHEC? Sorbitol MacConkey aka SMAC

Differential Plate Diagnosis Is SMAC selective or differential? Both!!!!

Differential Plate Diagnosis What grows on SMAC? Only allows gram (-) to grow

Differential Plate Diagnosis How do you read a SMAC agar? Normal flora ferment sorbitol -> RED EHEC doesn’t ferment sorbitol -> clear (looks green like gel)

Differential Diagnosis When do you most suspect rotavirus? Winter, in young children Test with immunoassay for Ag in stool

Differential Diagnosis How do you test for C. Difficile? Test for toxin A or B

Lab tests What is catalase used for? Differentiates between staph (+) and strep (-)

Staining How do you read an acid-fast stain ? Positive is pink (holds onto fuscin stain) Negative is blue (will absorb methylene blue stain)

Tuberculosis What are the symptoms of TB? Productive cough, chest pain, hemoptysis, and possibly cachexia

Tuberculosis How to diagnose TB? Presumptive- Skin test and chest X ray with acid-fast smear of sputum Definitive- Sputum culture, NAAT

Legionnaire’s Disease What are the symptoms of Legionnaire’s disease? Sudden onset cough, fever, chest pain Consolidation in lungs visible with chest X ray

Legionnaire’s Disease How to diagnoses Legionnaire’s? Direct fluorescent Ab – specific but not sensitive Urinary Ag test- Sensitive and specific!!

Legionnaire’s Disease Why can’t you stain or culture Legionella? Doesn’t hold stain and takes 3-5 days to culture -can be overgrown by other flora in specimen…

Differential Plate Diagnosis HEA plate with clear (green) colonies? Shigella

Differential Plate Diagnosis HEA plate with clear (green) colonies and “salmon eyes”? Salmonella…remember? H2S

Differential Plate Diagnosis HEA plate with Yellow/Brown colonies? Lactose positive colonies -> normal flora

Differential Plate Diagnosis Sorbitol Mac plate with red colonies? E. Coli!

Differential Plate Diagnosis Sorbitol Mac plate with clear colonies? E. Coli O157:H7 Remember...it doesn’t ferment sorbitol but the other E. coli do

Differential Plate Diagnosis Campy blood agar with growth? Duh, it’s Campylobacter

Differential Plate Diagnosis Campy blood agar with growth? Duh, it’s Campylobacter

Differential Plate Diagnosis What is TCBS agar? Dark-green sucrose plate

Differential Plate Diagnosis What grows on TCBS? Yellow -> Vibrio Cholera Green -> Vibrio parahaemolyticus

Diagnosis A child under five, gets diarrhea in the winter. What should you suspect besides bacteria? Rotovirus Test with immunoassay

Antibiotic Susceptibility What is the MIC? The minimum concentration (ug/mL) needed to inhibit bug growth Property of the bacteria...

Antibiotic Susceptibility What if the MIC is below susceptible MIC breakpoint? Susceptible

Antibiotic Susceptibility What if the MIC is above the resistant breakpoint? Resistant

Antibiotic Susceptibility What if the MIC is between the two breakpoints? Intermediate

Antibiotic Susceptibility What is Quantitative Susceptibility Testing? Take a pure colony. Match the turbidity to a standard. Add a little to different wells with diff. concentrations of Abx. It will tell you the concentration that inhibits growth

Antibiotic Susceptibility What is Qualtitative Susceptibility Testing? Use agar disk with uniform colonies and add antibiotic disks. Grow overnight. Measure inhibit of growth and compare to standard. Report either S, I or R (Lower MIC -> Bigger circle)

Antibiotic Susceptibility What kinds of cases should you use Quantitative MIC testing rather than qualitative? CNS issues, endocarditis, serious infections or slow-growing infections. ie, BAD STUFF

Sterile Body Sites What are the sterile body sites? Blood, CSF, pleural spaces, pericardial space, peritoneal space, synovial fluid, bladder -Anything cultured here is significant

Sterile Body Sites What is bacteremia? Bacteria in the blood stream

Sterile Body Sites What is septicemia? Bacteria dividing in the blood and associated w/ disease Must have 2 of the following: Fever, Tachycardia, Tachypnea, Abnormal WBC (high or low)

Sterile Body Sites How many blood cultures should be done? At least 3 over 24 hours, at least 1 hour apart.

Sterile Body Sites How is blood cultured? Take a lot (20mL) and add to enriched broth Let grow overnight Gram stain and culture

Sterile Body Sites Why can’t blood be gram stained? The volume on a slide may be too little to have any bacteria present on it.

Gram Negative Bacteria How can Gram (-) cocci be differentiated? N. Gonorrhea uses Glucose N. Meningitidis uses Glucose + Maltose N. Lactamica uses Glucose + Maltose + Lactose

Gram Negative Bacteria What is the significance of the oxidase test? Tests for Cytochrome Oxidase If (+) -> Pseudomonas (aeruginosa if Pyranin positive) If (-) -> Enterobacteriaceae (Use API to differentiate further)

Gram Positive Bacteria What is the significance of the Catalase test? Differentiates Staph from Strep Staph is (+) and Strep is (-)

Gram Positive Bacteria What is the significance of the coagulase test? Differentiates Staph aureus from CoNS Staph is (+) and other staph are (-)

Gram Positive Bacteria What is the significance of the hemolysis test? Alpha hemolysis -> Think Strep Ds and Strep pneumoniae Beta hemolysis -> Think Strep A and B (pyogenes and agalacticae)