Quiz 2: Lab Flashcards
antibiotic
a chemical synthesized by a microbe that will kill or inhibit other microbes
important producers of antibiotics
streptomyces, bacillus, penicillium, cephalosporium
tube dilution antibiotic sensitivity assay
a known concentration of antibiotic is diluted in a twofold tube dilution series and a drop of test organism is added to each tube to observe if good growth will occur. The antibiotic in the highest dilution tube that shows no culture growth is called the minimum inhibitory concentration (MIC).
kirby-bauer or disc method of determining antibiotic sensitivity
paper discs containing antibiotic are used to test for inhibition of bacterial growth in the vicinity of the disc on the agar plate. For standardization of the technique, a type of agar called Mueller-Hinton is used for plates. To determine if an isolate is resistant or sensitive to an antibiotic, the growth inhibition zone diameter (including disc diameter) is compaired with values produced by reference organisms of the same species on a standard table. Two antibiotics that produce the same inhibition zone diameter do diffusion rates effect results.
organism used for antibiotic disc assay
e. coli & staph. epidermidis
methods
Mueller-Hinton agar was used
Swabbing in 3 planes
disc assay: CAR 100
concentration: 100
inhibition zones:
e. coli: 2mm
s. epidermidis: 1.75mm
disc assay: C30
concentration: 30
inhibition zones:
e. coli: 2.25mm
s. epidermidis: 2.5mm
disc assay: Te30
concentration: 30
inhibition zones:
e. coli: 1.75mm
s. epidermidis: 0mm
disc assay: K30
concentration: 30
inhibition zones:
e. coli: 1mm
s. epidermidis: 1.5mm
What is the concentration of the antibiotic disk?
It is the number printed on the disc in units or micrograms
ADA: Did the two organisms tested show the same antibiotic sensitivity pattern? Why
No because bacteria reacts differently to different antibiotics
E.coli: thick peptidoglycan
S. epi: thin
Minimum Inhibitory Concentration (MIC)
least amount of antibiotic to stop bacteria
What is a bactericidal antibiotic?
antibiotic that kills microorganisms
What is a bacteriostatic antibiotic?
antibiotic that prevents growth
Explain the function of penicillinase
hydrolyzes penicillin
How are plasmids associated with the efficacy of antibiotic therapy?
They decrease the efficacy of antibiotic therapy because they introduce genes that are resistance to the antibiotics.
zone size interpretation chart
Carbenicillin (CB-100/CAR-100): disc potency: 100 ug – resistant: < 19 ; intermediate: 20-22 ; sensitive: >23
Chloramphenicol (C-30): disc potency: 30 – resistant: <12 ; intermediate: 13-17 ; sensitive: >18
Kanamycin (K-30): disc potency: 30 – resistant: <13 ; intermediate: 14-17 ; sensitive: >18
Tetracycline (Te-30): disc potency: 30 – resistant < 14 ; intermediate: 15-18 ; sensitive: >19
serum titer
the greatest dilution of serum which causes agglutination
acute serum
a sample taken from a patient at the onset of an illness
convalescent serum
a sample taken from a patient during recovery of an illness or several days after the onset
identification of etiological agent
if the microorganism being tested as an antigen results in the observation of an increase in convalescent titer at least 4 fold greater than the acute titer, it is likely to be the infectious agent
ATS: organism used & serums
sheep RBC (used as antigen)
serums:
phosphate buffered saline (PBS): 7.2 pH
NaCl: 8.77 g/Liter
KH2PO4: 5.10
Na2HPO4 7H2O: 10.50
What is titration measuring?
It is measuring the activity
When you are unable to measure concentration, what can you look for instead?
Instead, look for the activity AKA the titer of an antibody
Why are we unable to measure concentration for antibodies?
We cannot measure the concentration because we have antibodies for many things
ATS: explain the methods for the experiment
- placed PBS in each of the 9 tubes
- place 1 ml of the serum(aby) in position 1
- place .5 of serum in tube 2 which is one to two dilution
- take .5 from tube 2 and put in tube 3 this is one to four dilution
- continue two-fold dilution for all tubes and discard 0.5 ml from last tube
- add 0.5 of washed cells (sheep red blood cells -> antigen) to all 10 tubes
- incubate
- determine the titer
ATS: incubation standards
incubate the rack in 37 C for 1 hour
ATS: results
tubes 1-6: positive for agglutination
tubes 7-10: negative for agglutination
10 is control
tube number 6 (1/32) is the serum titer: tube number 6 is the last tube to show agglutination
What is serum titer normally?
1/16 or 1/32
What is agglutination?
When antigen combines with its antibodies causing clumping of cells
What happens in agglutination reaction when there is an excess of antibodies and what is it called?
Called prozone
Agglutination will not occur
Antibodies will bind to antigens univalently instead of multivalently
Antibodies that bind univalently cant crosslink one antigen to another
What occurs in agglutination when there is excess of antigens and what is it called
Agglutination will not occur
Called post-zone
Not enough aby and too many ag
No crossbridging
What is required for agglutination reaction to work optimally?
Specific ratio of aby:ag
AST: test question
antibodies cross between blood cells; network gets so big and falls out/agglutinates
How does an antibody affect a germ/bacteria?
- aby doesnt kill
- aby just signals
- wbc comes to kill
- complement binds and starts series of rxns
- complement proteins put holes in cell membrane
- water rushes into cell -> hemolysis
What creature makes the best complement?
guinea pig
What is the titer of someone who is vaccinated?
100
major illnesses caused by staphylococci
boils, scalded skin syndrome, toxic shock syndrome, food poisoning (staphylococcus aureus)
major diseases caused by streptococci
contagious skin disease, sore throat, rheumatic fever, glomerulonephritis (streptococcus pyogenes: the flesh eating bacteria); Pneumonia (streptococcus pneumoniae)
beta-hemolysis in staphylococci
this activity produces the lysis of red blood cells in agar plate medium. Blood agar is prepared by mixing 5% sterile defibrinated sheep blood with a nutritional blood agar that has been cooled to about 45 degree C after autoclaving. The medium is then dispensed into tubes or plates
hemolysis in streptococci
lysis of red blood cells in agar (beta-hemolysis), conversion of red blood cells to green (alpha-hemolysis), no effect on blood cells (gamma-hemolysis)
selective medium
medium to prevent growth of some species but permitting others to thrive
differential medium
medium that enables the investigator to distinguish one species from another. This is usually done through use of a chemical reaction which causes the color of the medium or colonies to change
BCGPC: organisms used
s. aureus
s. epidermidis
s. pyogenes
s. pneumoniae
s. fecalis
g. tetragena
BCGPC: major types of test media
for staph: mannitol salt, 110 agar, citrated rabbit plasma
for strep: bile esculin agar, taxos A disc, taxos P disc
agar slant: s. aureus (pathogen), s. epidermidis(normal flora), and s. faecalis (normal flora)
broth culture: gaffkya tetragena (normal flora)
BCGPC: What is the most dangerous organism?
s. pyogenes
BCGPC: gram stain results
S. aureus: gram positive, coccus, cluster
S. epidermidis: gram positive, coccus, cluster
S. faecalis: gram positive, coccus, chain
G. tetragena: gram positive, coccus, tetrad
BCGPC: catalase reaction
S. aureus: +
S. epidermidis: +
S. faecalis: -
G. tetragena: -
BCGPC: staphylococci
S. aureus: positive for all tests (hemolysis on blood agar, mannitol fermentation, gelatinase, coagulase)
S. epidermidis: negative for all tests(hemolysis on blood agar, mannitol fermentation, gelatinase, coagulase)
BCGPC: bile esculin azide agar
s. faecalis: good growth and blackening of medium
g. tetragena: no growth
Under the microscope, how can staphylococcus be differentiated from gaffkya tetragena?
Staph has grape-like clusters and g. tetragena is in tetrad
What is the chemical reaction mediated by the enzyme catalase?
2H2O2 > O2 + 2H2O
hydrogen peroxide to water and oxygen
What simple test can differentiate staph from strep?
catalase
Describe the mechanism by which staphylococcal coagulase causes plasma to clot?
conversion of fibrinogen to fibrin
-s. aureus produces bound & free coagulase (bound directly to fibrinogen)
major intestinal diseases
typhoid (salmonella typhi), dysentery (shigella dysenteriae), cholera (vibrio cholerae), travelers diarrhea (escherichia coli)
enterobacteria
gram-negative rods that are frequently found in the intestinal tract
facultative anaerobe
a bacterium that can grow in the anaerobic intestinal tract but also in the presence of oxygen
selective medium
medium to prevent growth of some species but permitting others to thrive
differential medium
medium that enables the investigator to distinguish one species from another. This is usually done through use of a chemical reaction which causes the color of the medium or colonies to change
macconkey agar
lactose fermenting organisms produce red colonies on this plate medium. Gram-positive bacteria are inhibited. The ingredients include lactose, bile salts and neutral red (pH indicator)
XLD
organisms that ferment the constituent carbohydrates produce yellow or red colonies on this plate medium. If lysine decarboxylase is produced colonies are red. Colonies may be black if H2S is produced. The medium contains xylose, lactose, sucrose, phenol red (pH indicator), bile salts, lysine, and ferric ammonium citrate (H2S detection)
TSI
triple sugar iron agar is used in the form of a slant tube. If an organism can use lactose or sucrose, the entire tube will turn yellow (acid). Gas may push the agar up the tube or form large bubbles. If only glucose is used, the base (butt) of the agar slant will turn yellow but the slant itself will remain red. An organism that produces H2S will cause the formation of a black pigment in the tube. Besides the sugars, the medium containts phenol red (pH indicator) and iron salts (H2S)
Simmons Citrate
this medium will turn blue if inoculated organism can use citrate. The medium includes citrate and thymol blue (pH indicator) and is prepared as an agar slant.
urea agar
organisms that can break down urea can be recognized by changing the color of this medium. Phenol red is used as an indicator.
iron Agar
this medium is for the detection of H2S produced by some species
Fermentation Broth
this is a test to determine if an organism can use a specific carbohydrate. The broth includes phenol red to test for acid produced from the carbohydrate.
A tiny inverted tube (durham tube) is present to assay production of gas
Decarboxylase Broth
this medium can detect the ability to produce decarbyoxylases by a color change of the tube
Motility Medium
if growth spreads from a straight vertical stab line of inoculation, the bacterium may be motile
enterotube II
this is a multiple test medium made by Roche diagnostics that can assay for several qualities at once
organisms used for biochemical characterization of enteric bacteria
e. coli
klebsiella pneumoniae
proteus vulgaris
pseudomonas aeruginosa
Try to describe in one or two sentences the basis behind identifying the species of a gram-negative rod
Why must the tube media be inoculated with a pure strain?
phage
this is a virus that infects bacteria
plaque
infective center (clear area) caused by phage on lawn of bacteria on a plate. It contains thousands of new phage. In this area the bacteria have been killled
plaque forming unit (PFU)
a phage particle that is capable of forming a plaque on a lawn of bacteria
methods the measure virus concentration
phage titration or plaque assay, routine test dilution (RTD), electron microscopy (EM), hemagglutinin titration, dose to infect 50% of subjects (ID50), dose to kill 50% of subjects (LD50)
PT: organism used
e. coli
remember for test
maximum is 10^11
ideal plaque count
30-300
What degree is agar melted at for phage titration experiment?
45 degrees C
For phage titration how do tubes get intubated?
36 degrees C for 20 minutes
For phage titration how to plates get intubated?
Upright position at 37 degrees C for 18-24 hours
Do you think every phage particle makes a plaque and why?
No, a cell infected by two phages makes 1 plaque because the phage grows as the cell grows
Some viruses and cells are dead
Why do the plaques stop growing?
There are mutants and they are no longer affected by the virus
Why can’t you see any plaques on zero dilution?
Plaques that overlap do not have a borders
What are those colonies on the zero dilution plate?
A few cells are resistant to infection
What does color change in the tube indicated?
positive inoculation
mycology
study of fungi
hypha
a filament of fungal cells
mycelium
mass of fungal hyphae
yeast
single celled fungi
pseudohyphae
short hyphal filaments consisting of rapidly dividing yeast cells
conidia
spores that develop on conidiophore
conidiophore
modified hypha for spore production in some fungi
fruiting body
large complex structure in some fungi to make spores
bud
offspring of yeast attached to parent cells
dimorphism
a characteristic of some fungi to exist in a yeast or hyphal phase depending on temperatiure
germ tube test
yeast cells of candida may produce short projections (germ tube) after inoculation into albumin and incubation for a few hours at 37 C
lacto-phenol cotton blue stain
a general purpose stain for examination of fungi
sabouraud dextrose agar
general purpose stain for the examination of fungi
aspergillus
a saprobe that can cause opportunistic infections of the lungs
candida albicans
a commensal organism that can be found on the skin, in the mouth, and in the genitourinary tract and causes opportunistic infections
basidiomycotina
group of fungi that includes edible mushrooms
mycology: organisms used
candida albicans grown at room temperature
candida albicans grown at 37 degrees C
apergillus nidulands grown at room temperature
mycology: structures from the specimens
hypha
mycelium
bud
conidia
conidiophore
pseudohyphae
germ tube
yeast cell
mycology: structures from the mushroom
cap
gills
ring
stipe
basidium
hymenium
what is the etiological agents and what are the major characteristics for each of the following diseases?
tinea corporis
histoplasmosis
coccidiodomycosis
parasite
an organism that lives on another to its detriment
trophozoite
adult vegetative form of a protozoan
cyst
small inactive state of protozoan
proglottid
a segment of tapeworm
entamoebba histolytica
pathogenic amoeba that causes severe dysentery and hepatic infection
giardis lamblia
flagellate that is a cause of weakness, abdominal pain and diarrhea
balantidium coli
ciliate that causes diarrhea, nausea, and vomiting
taenia saginata
beef tapeworm that inhabits the small intestine and is associated with weight loss and abdominal pain
enterbius vermicularis
pinworm (roundworm) that inhabits the colon and causes itching
fasciola hepatica
liver fluke that infects the bile ducts
parasitology: parasites examined
entamoeba histolytica
giardia lamblia
balantidium coli
taenia saginata (proglottid)
enterobius vemicularis (adult)
enterobius vermicularis (eggs)
fasciola hepatica
how do human beings become infected with the above organism?
