Microbiology Flashcards
Eukaryotic
contains genetic material within a membrane (nucleus)
Prokaryotic
genetic material but does not have a nuclear membrane
Ex. Bacteria
presumptive identification
your “best guess” on what is growing
gram stain
Primary stain - usually crystal violet
Mordant (fixes dyes to the cell wall) - Gram’s iodine solution
Decolorizer - 95% ethanol or acetone
Counterstain - basic fuchsin or safranin
gram positive
60-90% peptidoglycans in cell wall
Retain violet-iodine because of high # of peptidoglycans.
gram negative
10-20% peptidoglycans in the cell wall.
Lose crystal-violet w/ decolorizer and stains pink because low # of peptidoglycans.
gram variable
Gram variable implies that the bacteria will stain both purple and pink - but have all the same morphology (not the same as a mixed colony, where you can have cocci and bacilli together)
what causes gram variability
excessive decolorization overly thick smear excessive heat fixation old cultures poor quality stain (most commonly mordant - Gram’s iodine - being exposed to light
Potassium Hydroxide Test (KOH Test)
determines true gram status
Loopful of 3% KOH solution is placed on slide
Generous quantity of surface growth removed from culture and transferred to drop of KOH
Specimen stirred with loop the lifted gently and slowly. Gram positive bacteria mixture will remain homogenous and doesn’t lift with loop. Gram negative bacteria will develop mucoid appearance and produce a sticky strand when lifted.
Ziehl-Neelsen stain
Primarily used to ID acid-fast organisms of Mycobacterium and Nocardia species. 3 part stain:
Primary Stain - RED Acid-alcohol decolorizer Counterstain - BLUE Air dry - heat-fix - flood the slide with RED heat it until it steams - cool for 5 mins - rinse with tap water - acid alcohol decolorizer for 1-2 minutes (if negative, washed the RED away - if an acid-fast bacteria, it keeps the RED) - counterstain with blue - rinse - dry - examine Acid-fast = red Non-acid fast = blue
Giemsa stain
Used to detect spirochetes and rickettsiae as well as to demonstrate the capsule of Bacillus anthracis and the morphology of Dermatophilus congolensis
Baccili
Shaped like rods/cylinders
Cocci
Spherical cells
Spirochetes
Usually occur singly and can be subdivided into loose spirals, tight spirals, and comma-shaped spirals
pleomorphic
Shapes ranging from cocci to rods
arrangements
- Single - Spirilla & most Bacilli
- Pairs - Streptococcus pneumoniae (diplococcus)
- Chains - Short/Long Streptococcus spp.
- Tetrads - “quads”
- Clusters - Staphylococcus aureus (grapelike)
- Palisades - “Chinese letter” pattern Corynebacterium spp
spores
Some genera of bacteria produce endospores. Resistant to heat, desiccation, chemicals and radiation. Location in cells helps classify.
Obligate aerobes
Bacteria that require oxygen to survive.
obligate anaerobes
Bacteria that are killed in the presence of oxygen or those with growth that is inhibited in the presence of oxygen.
Facultative anaerobes
Organisms that can survive in the absence of oxygen, but their growth is limited.
Microaerophilic
Bacteria that prefer reduced oxygen tension.
Capnophilic
Bacteria that require high levels of carbon dioxide.
Fastidious microbes
picky eaters
general purpose media
Isolation of a wide variety of bacteria.
enriched media
Contains nutrients required to support the growth of a wide variety of bacteria - Meets the requirements of fastidious pathogens.
selective media
Allows certain types of bacteria to grow and inhibit growth of others.
differential media
Biochemical reaction to differentiate closely related organisms or groups of bacteria
enrichment media
Favors the growth of a particular type/group of organisms.
transport media
Keeps the sample as close as possible to its original state.
blood agar
Is an enriched medium, used to grow a wide variety of bacteria and a differential medium based on hemolysis.
hemolysis
Alpha Hemolysis- Partial hemolysis
Beta Hemolysis - Complete hemolysis, creates a clear zone around the colony.
Gamma Hemolysis - Hemolysis that produces no change in the appearance of the medium and around colonies.
Delta Hemolysis - Double zone hemolysis.
MacConkey agar
Is a selective (only grows Gram -) and differential medium (lactose fermenting = pink and non-lactose fermenting = no color change).
EMB eosin methylene blue
Is a selective and differential medium used to isolate fecal coliforms -
Allows some gram positive bacteria to grow. Escheria coli and Klebsiella pneumoniae = metallic green colonies
Thioglycollate broth
Is an enrichment broth used to determine oxygen requirements of microorganisms.
Mueller-Hinton Agar
Is a general purpose medium used for culture and sensitivity (susceptibility) testing of rapid growth bacteria.
culture incubation
Plates should be incubated at 37°C.
incubate upside down
To lessen contamination risk from airborne particles landing on them and to prevent water condensation
temperature
· Mesophiles- grow in moderate temperature.
· Psychrophiles- grow at a lower temperature.
· Thermophiles- grow in a higher temperature
bacteria produce
Binary fission
4 phases of growth for bacteria
Initial/Lag phase - Adjust to the environment.
Exponential - Increase in number of living bacterial cells, until space is used up or wastes accumulate.
The stationary phase - Plateau in number of living bacterial cells.
The death/final phase - Exponential decrease of living cells (spores are produced at this stage in sporulating species).
motility tests
wet prep
hanging drop
motility media
brownian movement
The random motion of particle as a result of collisions with surrounding gaseous molecules
why use motility test
If organisms are nonmotile on microscopic examination.
Catalase Test
Used to determine whether a gram-positive cocci is a staphylococci or streptococci
Positive- Bubbles after adding 3% hydrogen peroxide to slide. (staphylococci)
Negative- No bubbles. (streptococci)
Coagulase Test
This test is done to differentiate among coagulase-positive Staphylococcus aureus, Staphylococcus pseudointermedius and coagulase-
negative Staphylococcus (e.g. Staphylococcus epidermidis or Staphylococcus saprophyticus)
slide coagulase
Commercially available. Detects surface-bound coagulase or clumping factor.
Mix a loopful of staphylococci from a colony in a drop of water/saline solution to yield a thick suspension. Add a drop of fresh rabbit/human plasma and stirred with a sterile loop. Clumping within 5-20 seconds indicates a positive reaction.
tube coagulase
Uses lyophilized plasma that has been diluted according to the manufacturer’s directions. Inoculate a loopful and culture with noninhibitory medium, such as blood agar. It is incubated at 37°C and read hourly for 4 hours.
Positive- Clot formation. (Staphylococcus aureus)
Negative- No clot formation. Note: If test results remain negative, the sample is incubated again for 24 hours and then read. (non-pathogenic Staphylococcus spp.)
why coagulase test
Catalase-positive, gram-positive cocci
Which enzyme coagulates plasma? Coagulase
oxidase activity
The oxidase test identifies organisms that produce the enzyme cytochrome oxidase. Escherichia coli vs Pseudomonas aeruginosa
Positive = PURPLE - Microorganisms are oxidase positive when the color changes to purple within 15 to 30 seconds. Pseudomonas aeuriginosa
Negative- = No color change Escherichia coli
common bacteria
Streptococcus spp. = Gram positive cocci- catalase negative
Staphylococcus epidermidis = Gram positive cocci - catalase positive - coagulase negative
Staphylococcus aureus “golden staph”= Gram positive cocci - catalase positive - coagulase positive
Pseudomonas aeruginosa = Gram negative bacilli - oxidase positive
Escherichia coli = Gram negative bacilli - oxidase negative
acid production for glucose
A tube of 1% glucose in peptone broth that contains a pH indicator is inoculated and incubated for 24-48 hrs at 37℃.
For fastidious organisms- appx. 5 drops sterile serum should be added to the peptone water medium, or growth may not occur.
California mastitis test
Helps confirm the presence of subclinical mastitis. By using milk, a reagent, and a paddle
staining milk
Prepare a thin smear of mastitic milk. The smear is heat fixed and stained with gram stain or methylene blue.
agar diffusion method
Most commonly performed sensitivity test. Uses paper disks impregnated with antibiotics. Quantitative tests measure inhibitory zones to estimate antimicrobial sensitivity. (FDA method, Kirby-Bauer, International Collaborative Study methods)
Kirby-Bauer Method
Most commonly used method. Uses paper disks impregnated with antimicrobials. Quantitative and requires the measurement of ZOI, which gives an estimate of susceptibility.
minimum inhibitory concentration
Lowest concentration of the specific antimicrobial that can inhibit the growth of a given bacteria
antimicrobial disks
It is paper with known concentrations of antibiotics
direct sensitivity testing
Application of undiluted samples directly to Mueller Hinton plate. Not as precise as indirect testing, reasonable results when only one organism present
zone of inhibition
area of no bacterial growth around an inhibitory disk that indicates some sensitivity of the particular organism to an antimicrobial.
reading ZOI
Should be read after a constant period, most satisfactorily after overnight incubation - 18 to 24 hours. Rapid results may be read after 6 to 8 hours of incubation. The diameter of each inhibition zone is measured from the underside of the plate using calipers, a transparent ruler, or template. Zones are measured and recorded to the nearest millimeter.
Note: For Mueller-Hinton agar with blood, the zone size must be read from the top surface, with the lid of the plate removed.
heterotrophs
Digest complex organic material (plant/animal) food internally - enzymes. May be Parasitic or Saprophytic.
parasitic
Cause harm to the plant/animal host it is using for food
saphrophytic
Feed on dead/dying material - These are the fungi you see on fallen trees.
yeast
Single-celled and reproduces by budding.
hyphae
Multicellular fungal organism - Microscopic feeding form
mycelium
Large branched webbings. Composed of hyphae
Sabouraud Dextrose
DTM commonly found in most clinics, contains an indicator that turns red if dermatophytes are present
collect dermatophytes
Pluck: To pull hair out along with the fungus with forceps/tweezer.
- Tooth brush: Obtain a new toothbrush. Brush suspected lesion for 1 to 2 minutes.
lactophenol cotton blue
ID fungal organisms
Microsporum canis
Little boats with pointed ends.
Microsporum gypseum
Little boats with blunted ends.
Trichophyton mentagrophytes
Little coffins with circular friends
dermatophyte testing
Wood’s lamp: warm up light for 5 minutes. Infected hairs will glow in a dark room using fluorescence light.
Dermatophyte test medium (DTM): Culture media will turn red in the presence of most dermatophytes.
Rapid sporulation medium (RSM)/ Enhanced sporulation medium (ESM): Culture media will turn Teal in the presence of most dermatophytes. What color does a positive result for DTM yield? Red If the color change occurs, is that 100% positive? No. Confirm with microscopic examination.
non dermatophytes
Bismuth-Glucose-Glycine-Yeast (Biggy), Blood agar or Sabouraud dextrose agar
microscopic evaluation of fungi
Collect mycelium from media with sticky side of clear cellophane (Scotch) tape, place a strip of lactophenol cotton blue (or methylene blue from DiffQuik) onto a slide and place the tape onto the stain - examine on 10x and 40x for specific spores (tells you the organism)
send out viral samples
Check with the diagnostic laboratory for a preferred sample and necessary transport medium.
cell culture
need living cells to grow and replicate, Animal’s cells grown in vitro, continuous cell lines and single cell type. Fetal kidney, embryonic trachea, skin from lab animals. Inoculated into primary culture from the same species from which specimen was taken.
Immunologic and Molecular Diagnostics Examination
Definitive ID requires serologic procedures, in-house tests for viral pathogens, Molecular testing, PCR also used to ID pathogens.
Viruses are often present in nasal or pharyngeal secretions in early stage of URI (Upper Respiratory Infection). Mucosal scrapings rather than swabs of the secretions should be taken. Viral diseases are often complicated by pathogenic bacteria (secondary infections), which can turn into a mild viral infection into a serious disease
histopathology
For viral samples – thin tissue sections placed in 10% formalin – no freezing – may also do 2x2 inch cubes that contain normal and abnormal tissue
submission virus samples
refrigerated at 4°C. Why? Viral titers decrease as temperature increases.
- If delivered within 24 hours, what do you do? Pack with coolant packs or ice in polystyrene-insulated carton for shipment
- If over 24 hours, what do you do? Snap freeze at -70°C and ship on dry ice. However, suspected parainfluenza and influenza viruses being shipped are best preserved at -20°C.
- Small tissue pieces, fecal material, or mucus preserved in vials filled with 50% glycol and stored at 4° C
- Scanning electron microscopy
a. Fixative- 10% buffered neutral formalin added to sample at maximum fixative-to-sample ratio 1:1. - Urine samples
a. 5 mL in sterile container
b. Chilled within 24 hours or frozen
