Clinical micro lab exam 1

BSL 1

not known to cause disease in healthy adults

BSL 2

indigenous microorganisms that can lead to disease in healthy adults

BSL 3

indigenous or exotic microorganisms that can cause serious illness through respiratory transmission

BSL 4

Exotic microorganism that are aerosol transmitted. Rarely have treatments and are often fatal

Dark field microscope

used for living cells that would be distorted by heat drying or staining

phase contrast microscope

useful for observing intracellular structures and locomotor structures (cilia)

Fluorescence microcope

useful in diagnosing infections and pinpointing particular cellular structures

Confocal microscope

most often used for fluorescently stained specimens

TEM microscope

best for viewing detailed structure of cells and viruses

SEM microscope

shows a 3D view of outside of organism

acid fast stain

acid-fast organisms stain pink and non acid fast stain blue

useful for mycobacterium tuberculosis

selective media

only allow growth of particular organisms

Differential media

distinguish among different groups of microbes

Endospore stain

endospores are green

vegetative cells are pink/red

useful for Bacillus anthracis, Bacillus cereus, and Clostridium species

Acridine Orange Stain

differentiates RNA and DNA

bacteria appear orange

Host cells are green

Cary Blair TM

stool and rectum swabs

Shigella, Salmonella, Vibrio cholerae, E.coli

Stuart TM

used for fastidious organisms

BACTEC, BacT/ALERT, VersaTREK

blood culture tubes that monitor CO2 production to flag any growth for common pathogens

positive bottle is not an identification, further steps required

Chromogenic Agar

contain enzyme substrates associated with a specific target organism

if enzyme is present, color will show

Automated biochemical systems (VITEK 2, BD Phoenix, Micro Scan)

inoculate cards or panels with preloaded substrates and computer reads color changes, can also read susceptibility

MALDI-TOF Mass Spectrometry

laser ionizes sample and reads protein fingerprint of organism

does not test for susceptibility

Syndromic Multiplex PCR

bedside PCR that looks for DNA of a panel of organisms

Nucleic acid amplification test (NAATs)

real time PCR that uses multiplex syndromic panels that test many pathogens and selected resistance genes

Probe hybridization

detect specific sequences of DNA

good for culture ID confirmation

rapid ID from positive blood cultures

sequencing

used when culture/biochemical ID fails or for epidemiology

16s rRNA sequencing

gives ID to genus level

helpful for when MALDI-TOF or biochemical tests cannot identify

Whole genome sequencing

current standard for outbreak investigations

gives species when MALDI-TOF or 16s sequencing is inconclusive

Metagenomic Next Generation Sequencing

“shotgun” approach

sequence millions of short DNA fragments

genus ID

Antigen Detection

detect pathogens quick when a culture may take too long or organism is difficult to culture

used in strep swabs

Minimum inhibitory concentration

lowest concentration of an antimicrobial that prevents visible growth

Minimul lethal concentration (MLC/MBC)

lowest concentration that kills all bacteria

bacteriostatic

inhibits growth

bactericidal

kills organisms

Etest method

strip with increasing levels of antimicrobials

can show MIC but not MLC

Staphylococcus catalase

positive

streptococcus catalase

negative

Staphylococcus aureus Gram and arrangement

Gram positive cocci in clusters

what is MRSA resistant to

methicillin (Cefoxitine)

cause of scalded skin syndrome

Staphylococcus aureus

Alpha hemolysis

partial breakdown

green color

Beta hemolysis

complete hemolysis

clearing around the colony

Gamma hemolysis

no hemolysis

Staphylococcus epidermidis Gram and morphology

Gram + cocci in clusters

causes of impetigo

Staphylococcus aureus and Streptococcus pyogenes

Streptococcus pyogenes Gram and morphology

Gram positive cocci in pairs or chains

cause of necrotising fasciitis

Streptococcus pyogenes

Enterococcus faecalis Gram and morphology

Gram positive cocci in pairs or chains

Corynebacterium jeikeium Gram and morphology

Gram positive rods

form V or Y shapes

Clostridium tetani Gram and morphology

Gram positive rods

Clostridium tetani spore formation

form “drumstick” spores

Clostridium perfringens Gram and morphology

gram positive rods

positive reverse CAMP

Clostridium perfringens

Gas gangrene

Clostridium perfringens

Clostridium perfringens lecithinase

positive

Bacillus antracis Gram and morphology

Gram positive rods in chains

Bacillus antracis spore formation

forms spores but not always seen

Bacillus antracis lecithinase

negative

Erysipelothrix rhusiopathiae Gram and morphology

Gram positive rods

Acinetobacter baumannii Gram and morphology

Gram negative rods

Acinetobacter baumannii EMB

clear with blue-gray colony centers

Pseudomonas aeruginosa Gram and morphology

Gram negative rods

Pseudomonas aeruginosa odor

grape like odor

E. coli, Salmonella, & Proteus mirabilis Gram and morphology

Gram negative rods

E. coli EMB

metallic green

Salmonella H₂S

positive

P. mirabilis motility

swarming motility

Pasteurella multocida Gram and morphology

Gram negative cocci/rod

Cat and dog bites/scratches

Pasteurella multocida

Klebsiella pneumoniae Gram and morphology

Gram negative rod

surgical incisions and wounds

Klebsiella pneumoniae

Vibrio vulnificus Gram and morphology

Gram negative curved rods

Gulf waters and raw/undercooked seafood

Vibrio vulnificus

Moraxella catarrhalis Gram and morphology

Gram negative cocci: single, pairs, or tetrads

Aerobe

requires oxygen to grow

Anaerobe

oxygen is detrimental to organism

Mircroerophile

organism requires small amounts of oxygen to grow

capnophile

organism requires high concentrations of CO₂ to grow

Bile esculin hydrolysis

positive: blackening of slant

Catalase

positive: bubbles

EMB

Acid ++: green

Acid +: purple

Acid -: pink or colorless

Gamma-glutamylaminopeptidase

Neisseria test

Yellow: Neisseria meningitdis

Red: Neisseria gonorrheaea

Hippurate hydrolysis

Positive: blue

Negative: no color change

KOH

positive: string present (gram negative)

negative: no strings (gram positive)

 

MTM (Modified Thayer-Martin) agar

selective for Neisseria

MSA (Mannitol Salt Agar)

Acid +: yellow

Acid -: Red

PR tubes

positive (acid production): yellow

negative: red

PYR disk

positive: red

negative: no color change

Indole spot test

positive: blue

negative: red

SIM

Hydrogen positive: black

Indole positive: red after addition of kovacs

Motility positive: cloudy appearance outward from the stab line

swarming motility

growth all over plate, no clear isolation of colonies

Urea

positive: pink

negative: orange/yellow

X and V factors

differentiate Haemophilus spp and Haemophilus influenzae

Group A streptococcus

Streptococcus pyogenes

CAMP test is used to identify

Listeria monocytogenes and Streptococcus agalactiae

CAMP test

B hemolytic Staphylococcus aureus is streaked down center

organism is streaked perpendicular and to almost touching center line

Reverse CAMP tests for

Clostridium perfrinfens

Reverse CAMP test

Streptococcus agalactiae is streaked down center

incubated anaerobically

KOH test purpose

confirms gram stain on wether KOH can lyse bacterial cell wall and release DNA

Nitrate reduction steps

inoculate nitrate and incubate

add nitrate A and B

if no color change add zinc

Nitrate results

Red color after adding A and B: bacteria reduced nitrate to nitrite

Red color after zinc: failed to reduce nitrate

No color change: nitrate to nitrogen gas or ammonia