Lab Practical Final - Experiments/Exercises Flashcards
Kirby-Baur test…
determines which antibiotics are effective against a given bacterium
Resistant:
antibiotics will have no effect on bacteria growth (they don’t work)
Susceptible:
antibiotics work; bacteria is sensitive to antibiotics
Zone of inhibition:
area of no growth
A chart must be used to read the results because…
the zone of inhibition varies for each antibiotic
Determining antibiotic effectiveness.. Disk potency: Resistant: Intermediate: Susceptibility:
Disk potency: measurement of antibiotic strength
Resistant: ability of the microbe to grow in the presence of antibiotic
Intermediate: bacteria falls short of resistance
Susceptibility: antibiotics work
Antibiotic:
substances that kill or prevent growth of bacteria
Broad spectrum:
Narrow spectrum:
Why should one be used over the other?
Broad: acts against wide range of bacteria
Narrow: acts against specific bacteria
Broad spectrum may attack normal microbiota and immune system (acts against Gram + and Gram -)
Bacteriostatic:
Bactericidal:
Bacteriostatic: slows/stops bacteria growth
Bactericidal: kills bacteria
Disinfectant:
used to kill bacteria
We determined which disinfectant worked best by…
using the spread technique with 2 plates (E. coli and S. aureus) and placing paper disks dipped in disinfectant on those plates
The effect that pH has on bacterial growth…
set of cardinal pH: minimum growth pH, maximum growth pH, optimum pH (grows best)
Acidiophiles:
Neutrophiles:
Alkaliphiles:
Acidiophiles: pH below 5.5
Neutrophiles: pH 5.5-8.5
Alkaliphiles: pH above 8.5
The effect temperature has on bacterial growth…
set of cardinal temperatures: minimum, maximum, optimum (highest growth rate)
Psychrophiles: Psychrotrophs: Mesophiles: Thermophiles: Extreme thermophiles:
Psychrophiles: grow 30˚C
Mesophiles: grow in moderate temps 15˚C - 45˚C
Thermophiles: grow in warmer temp >40˚C
Extreme thermophiles: grow in high heat 60-110˚ C
Aerotolerance:
refers to the ability of an organism to live and grow in the presence of oxygen
The medium used to test aerotolerance:
- a fluid thioglycollate medium: contains an oxygen gradient ranging from anaerobic at the bottom of the tube to aerobic at the top
- the chemical resazurin is included in the media to indicate the presence of oxygen by producing a pink coloration where oxygen is present
Obligate aerobes:
Facultative aerobes:
Aerotolerant anaerobes:
Obligate anaerobes:
Obligate aerobes: require oxygen for respiration; grow at top
Facultative aerobes: presence OR absence of oxygen; everywhere but more at top
Aerotolerant anaerobes: do not require oxygen but are not harmed by it; grow everywhere
Obligate anaerobes: cannot grow in the presence of oxygen; grow at bottom
Methylene blue reductase test reaction…
- methylene blue dye is blue when oxidized and colorless when reduced
- good quality milk takes longer than 6 hours to convert the methylene blue
- blue clears faster = more microbes
Water quality is determined with such extensive tests because…
- there are 3 groups (with 5 tubes) with different strengths
- the greater the # of tubes per group, the greater the sensitivity of the test
- development of gas in any tubes is evidence of the presence of coliform bacteria in the sample
Coliform bacteria…
- Gram negative
- Bacillus
- Non-sporulating
- Rapid lactose fermenter; acid and gas
3 tests done to detect presence of coliform bacteria:
- presumptive (test tubes)
- confirmed (EMB)
- completed (not done in lab)
Lactose broth and inverted Durham tubes…
- in the presumptive test
- lactose: bacteria are capable of using lactose as carbon source
- inverted Durham tube: gas collection
MPN:
- Most probable number test
- estimated by determining the number of tubes in each group that show gas following the inoculation period
EMB plates is used in the confirmed test because…
- eosin-methylene blue contains the dye methylene blue, which inhibits growth of gram + organisms (in the presence of an acid environment, EMB forms a complex that precipitates out into the coliform colonies, producing dark centers and a green metallic sheen; characteristics of E. coli, major indicator of fecal pollution)
- Endo agar is a nutrient medium containing the dye fuchsin, which is present in the decolorized state (in the presence of acid produced by the coliform bacteria, fuchsin forms a dark pink complex that turns the E. coli colonies and the surrounding medium pink)
Purpose of the completed test…
- the final analysis of the water sample
- used to examine the coliform colonies that appeared on the EMB or end agar plates used in the confirmed test (an isolated colony is picked from confirmatory test plate and inoculated into a tube of lactose broth and streaked on a nutrient agar slant to perform a gram stain)
- positive (presence of E. coli) = acid and gas in lactose broth and the presence of gram negative bacilli
Why do we look for E. coli when using water potability?
Presence of E. coli = positive confirmed test (water is non-potable)
Absence of E. coli = negative (water is not contaminated)
Why is an EMB plate used in the food analysis experiment?
TFTC = less than 30 TMTC = over 300
Why did we use 3 different agar pour plates in the food analysis experiment?
3 different dilutions:
10^-2
10^-3
10^-4
Is the presence of microbes a definite indicator that the food is unsafe?
No; some food needs microbes
What did we use to differentiate staphs from streps?
Catalase test: identifies organisms that possess the enzyme catalase (breaks down hydrogen peroxide into water and gaseous oxygen)
- Staph = catalase-positive; bubbles form
- Strep = no catalase; no bubbles
How to differentiate staph and strep…
Staph: cocci, clusters (Gram +)
Strep: cocci, chains (Gram +)
Tests used on Staphs…
Coagulase test
DNase test
Mannitol Salt Agar (MSA)
Novobiocin Sensitivity test
Coagulase test
- enzyme used by Staph aureus to cause formation of blood plasma clots around themselves, mechanism of evading hosts’ immune system defense
- looks for positive indiction of Staph aureus
- Identifies a Staph aureus by identifying the presence of the enzyme coagulase
DNase test
- DNase agar is a method to distinguish Staph aureus from Staph saprophytic and Staph epidermidis
- Staph aureus contains DNA bound to methyl green dye
- If organism excretes DNase, DNA is hydrolyzed into smaller pieces and methyl green leaves a clearing around bacterial growth (positive)
- Identifies Staph aureus by identifying the presence of the enzyme DNase
Mannitol Salt Agar (MSA)
- Both selective and differential media
- Contains 7.5% salt, which prevents growth of most organisms
- Staph does WELL in high salt; prevents growth of most organisms
- The carbohydrate mannitol is used to differentiate between the mannitol fermenting pathogenic Staph and the non-mannitol fermenting staph
- Fermentation of mannitol = produces acid (phenol red turns yellow)
- Identifies if bacterium is from the genus Staph and if a Staph is the mannitol fermenting Staph aureus
Novobiocin sensitivity test
- Uses disks impregnated with the antibiotic novobiocin
- Bacteria susceptible to antibiotic = will not grow well (Staph epidermidis and Staph aureus)
- Resistant to antibiotic = will grow (Staph saprophyticus)
Tests used on Streps…
Hemolysis
Bacitracin sensitivity
Bile esculin test
Hemolysis
- an enzyme that destroys or partially destroys red blood cells
- Beta-hemolytic strep: breaks down RBCs; looks like clearing around colonies (Strep pyogenes and Strep agalactiae)
- Alpha-hemolytic strep: partially breaks down RBCs; looks like green coloration around colonies due to hemoglobin metabolite of the unloosed RBCs (Strep pneumonia and sometimes Enterococcus faecalis)
- Gamma-hemolytic: no break down; no color change, just growth in colonies (sometimes Enterococcus faecalis)
Bacitracin sensitivity test
-Uses paper disk impregnated with the antibiotic bacitracin
-Susceptible to bacitracin = zone
-Resistant to bacitracin = growth around disk
-Useful in differentiating the beta-hemolytic group A Strep from the other beta-hemolytic Strep
(Step pyogenes is bacitracin susceptible)
Bile esculin test
- Bile esculin agar is used to detect Group D Strep and Enterococci
- Media contains bile which inhibits growth of organisms except Group D Strep and Entero
- They hydrolyze esculin into glucose and esculetin (glucose is used for energy metabolism and esculetin reacts with iron salts in the media to form black precipitate. Dark coloration = Group D Strep or Entero)
Staph aureus results... Catalase: Coagulase: DNase: Mannitol fermentation: Novobiocin:
- Catalase: positive = bubbles
- Coagulase: positive = solid
- DNase: positive = clearing
- Mannitol fermentation: positive = turns yellow
- Novobiocin: susceptible
Staph saprophyticus results... Catalase: Coagulase: DNase: Mannitol fermentation: Novobiocin:
- Catalase: positive = bubbles
- Coagulase: negative = not solid
- DNase: negative = green; no clearing
- Mannitol fermentation: variable
- Novobiocin: resistant
Staph epidermidis results... Catalase: Coagulase: DNase: Mannitol fermentation: Novobiocin:
- Catalase: positive = bubbles
- Coagulase: negative = not solid
- DNase: green; no clearing
- Mannitol fermentation: negative = red
- Novobiocin: susceptible
Strep pyogenes results... Catalase: Hemolysis: Bacitracin: Bile esculin:
- Catalase: negative = no bubbles
- Hemolysis: beta (destroy RBCs)
- Bacitracin: sensitive
- Bile esculin: negative = no black precipitate
Strep agalactiae results… Catalase:
Hemolysis:
Bacitracin:
Bile esculin:
- Catalase: negative = no bubbles
- Hemolysis: beta (destroys RBCs)
- Bacitracin: resistant
- Bile esculin: negative = no black precipitate
Strep pneumoniae results... Catalase: Hemolysis: Bacitracin: Bile esculin:
- Catalase: negative = no bubbles
- Hemolysis: alpha (partially destroy RBCs)
- Bacitracin: resistant
- Bile esculin: negative = no black precipitate
Enterococcus faecalis results... Catalase: Hemolysis: Bacitracin: Bile esculin:
- Catalase: negative = no bubbles
- Hemolysis: alpha (partially destroy RBCs) or gamma (doesn’t destroy RBCs)
- Bacitracin: resistant
- Bile esculin: positive = back precipitate
