Lab practical 2 part 2
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What is sucseptibility?
Bacterial growth is inhibited by concentrations of antimicrobial agent when the recommended dosage is used for the site of infection
What is resistance?
A bacteria's ability to ward off the effects of an antibiotic, disinfectant etc
Describe susceptibility vs resistance.
Susceptibility is the effect an antimicrobial agent has on an organism where as resistance is the ability of an organism to ward off an antimicrobial agent
How do we determine bacterial resistance or susceptibility?
- By utilizing a Standardized disk susceptibility test
- aka Kirby-Bauer method
Explain the Kirby Bauer Method.
- Bacteria swabbed onto Mueller-Hinton agar
- Paper discs w/known amts of antibiotic are added
- Plates incubated
- Plates observed for Zones of inhibition
___-___ agar gives reproducible results & does not inhibit sulfonamides
Why might colonies appear in a zone of inhibition?
May predict eventual resistance to that agent
What media are used to test for Staphylococcus and why?
Mannitol Salt agar b/c the high salt content allows staphylococcal growth but not others
Why is mannitol salt agar both selective and differential?
selective for gram positive bacterium Staphylococci since this level of NaCl is inhibitory to most other bacteria. It is also a differential medium for mannitol fermentors
What media do you need to test for Streptococcus?
Blood agar base such as trypticase soy agar w/defibrinated whole sheep blood
Why does blood agar select for Streptococcus?
The organism lacks the certain enzyme systems for nutrition therefore cultivation on a rich medium is necessary
Is mitis salivarious agar selective or differential? Why?
Selective b/c crystal violet, potassium tellurite & trypan blue which inhibit gram-neg bacilli and gram pos bacteria other than Streptococci
What does S. mitis (alpha-hemolytic, gram pos coccus) look like on mitis salivarious agar?
Flat, blue colonies w/dark centers
What does S. salivarious (gram pos sphere) look like on mitis salivarious agar?
Lg, blue dome shaped colonies w/a gumdrop appearance
What does S. mutans (gram pos coccus) look like on mitis salivarious agar?
Blue colonies having domed brown center
Gram-neg & non-streptococcal gram-pos bacterial growth is inhibited on ___ ___ agar.
Explain why throat culture samples are swabbed on Blood Agar.
Cultivation on a rich medium like blood agar is necessary b/c the organism lacks certain enzyme systems for nutrition
___ ___ streptococci on blood agar destroy RBCs in the medium completely & form clear zone around the colonies?
Alpha-hemolytic streptococci react how on blood agar?
- Incomplete destruction of RBCs
- Colonies surrounded by olive-green or brown discoloration of the medium
What is an example of a beta-hemolytic streptococci?
S. mitis (gram pos coccus) & S. pneumoniae (gram pos, alpha hemolytic) are 2 examples of ___ hemolytic streptococci.
____ streptococci cause no hemolysis of RBCs and an example is what?
- S. lactis the organism in yogurt
What do you use McConkey agar for?
To isolate enteric bacteria b/c it is a differential medium containing bile salts to inhibit nonenteric bacteria
What do you use EMB agar for?
To isolate enteric bacteria
Is MacConkey agar selective, differential or both and why?
- Differential b/c:
- It contains bile salts to inhibit nonenteric bacteria
- It contains Crystal violet dye & natural red dye
Is EMB agar selective, differential or both and why?
Differential medium that does not support the growth of gram pos bacteria
What is difference between enteric bacteria and coliforms?
Coliforms are collection gram-neg nonsporeforming rods that ferment lactose to acid & gas where as enteric bacteria
What happens to TSI agar if an organism ferments ONLY glucose?
- Slant & butt turn yellow
- Butt remains yellow
- Slant reverts to red as alkaline conds reappear
Interpret results: Sufficient acid is produced to cause both slant & butt of TSI agar both remain yellow
Lactose, sucrose or both carbs are being fermented
What can we conclude if the slant and butt of a TSI deep remain red alkaline color after incubation?
No carbohydrate has been fermented
What does TSI stand for?
- Triple sugar iron agar
- 3 carbs (glucose, lactose, sucrose)
- pH indicator Phenol red
- Presence of cracks & fissures indicate CO2 production
What does IMViC stand for?
- Indole Production
- Methyl red test
- Voges-Proskauer test
- Citrate test
Complete the indole test by adding 5gtts of ___ reagent to trypticase soy or tryptone broth tube.
What indicates a positive result for the Voges-Proskauer test?
Acetylmethylcarbinol reacts w/alpha naphthol & Potassium Hydroxide to form RED CHEMICAL COMPOUND
How id Voges-Proskauer completed?
Adding 10gtts of alpha napthol, mix, then add 10gtts of patassium hydroxide
What is Voges-Proskauer test?
Utilizes digestion of glucose to acetylmethylcarbinol to differentiate enteric species
Eval results for the citrate test?
- Pos - blue color indicating ability to use citrate
- Neg - Green color
What is the citrate test?
Done using Simmons agar and determines ability of certain bacteria to use citrate, a salt of citric acid, as a sole carbon source in growth
What indicates a + result for the Methyl Red test?
When added at end of incubation period methyl red remains red in presence of acid but becomes yellow in alkaline or neutral solution
Explain methyl red test.
- Determines an organisms ability to ferment glucose & produce lg amts of acid
- Completed by adding 5 drops of methyl red to MR-VP tube
How do we interpret an MPN test?
- We examine each set of inoculated tubes.
- Any tube w/at least 10% gas production is +
- Compare the number of + tubes w/an MPN index to determine # of coliform/100ml
What are you estimating w/an MPN test?
The number of coliform bacteria present in order to get an idea of the extent of bacterial polution
What steps follow a positive MPN test?
A confirmed test then a completed test
What conclusions may be drawn if no growth appears on MacConkey or EMB agar even though the MPN test was positive?
From this result we can conclude that human waste is present in the water however the bacteria are not classified as enteric as well
Give 4 examples of Bacillus species & a common use for each.
- B. subtillis (Gram pos rod) - Source of enzymes in enzyme detergents
- B. polymyxa (Gram pos) - antibiotic polymyxin
- Geobacillus stearothermophilus - tests effectiveness of sterilization
- B. anthracis (gram pos rod) - agent of anthrax
Give 4 examples of Clostridia species & what they cause.
- C. botulinum (gram pos rod) - causative agent of botulism
- C. difficile (gram pos rod) - causative agent of pseudomembranous colitis
- C. tetani (gram pos rod) - causative agent of tetanusC. perfringens (gram pos rod) - causative agent gas gangrene
Why did we heat the Bacillus samples but not the Clostridia samples?
B/c we wanted to destroy all organisms other than endospores. This was not required w/Clostridium b/c we were working in an anaerobic environment in which only clostridial species will grow thereby separating them out
What special procedure did we use to grow Clostridia?
We placed our plates inoculated with Clostridia in sealed bags in order to ensure an anaerobic environment
Growth of Clostridium species is generally accompanied by foul odors and blackening of the medium. What are the biochemical basis for these reactions?
This is due to the production hydrogen sulfide as a byproduct of digestion
A + result for Clostridia in thioglycolate medium is displayed by what?
- Cloudiness below the methylene blue indicator
- Gram stain reveals Gram-pos spore forming rods
How does a pos result for Clostridia in litmus milk agar appear?
The presence of Clostridia in cooked meat medium is indicated by what 3 characteristics?
- Meat particles turn to fine sediment
- Odor of hydrogen sulfide
- Blackening of the medium
How is the presence of Clostridia seen on brain heart infusion agar?
- Blackening of the medium
- Colony formation deep within the agar
The presence of Clostridia on SPS agar is indicated by the presence of ___ colonies.
Clostridium species are indicated on blood agar by the presence of a ___ ___ of ___.
Double Zone of Hemolysis
___ is a mass of fungal filaments or hypha that can be seen with the naked eye growing across top of the growth medium.
Molds grow as long, tangled filaments of cells and each individual filament is known as a ___.
What molds and yeasts did we use in class?
- Saccharomyces cerevisiaeAspergillus nigerPenicilium chrysogenum
What media are required to cultivate molds and why?
Sabouraud dextrose agar b/c of its acidity & the added carbohydrates are favored by molds
Could reliable results be obtained if TSI agar were inoculated w/a sample directly fr a fecal suspension?
No, b/c for the best results we would want to use an isolated colony rather than the actual sample which could be a mixture of many different things
Describe broad spectrum antibiotic.
One which works on a wide range of bacterial species
Describe narrow spectrum antibiotics.
Those which are limited in the bacteria it will effect
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