BIO1913 Lab Practical
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What are the 3 factors which would cause a Gram positive bacteria to stain like a Gram negative bacteria?
(The reverse RARELY happens)
- An OLD CULTURE: the culture should be 24-48 hrs. old
- Improper HEAT FIXATION: if the smear is heated too long or at too high a temp., it will weaken the cell wall structure.
- OVER DECOLORIZATION: if alcohol is applied for too long of a period, some Gram + cells will lose the crystal violet dye and counterstain with safranin.
List and explain the Reagents used in a Gram stain.
- Gram's Crystal Violet: Primary Stain.
- Gram's Iodine: Mordant - enhances and fixes the crystal violet in the cell wall.
- Alcohol: Decolorizer.
- Gram's Safranin: Counterstain.
What purpose does a SPORE serve?
Survival during hard conditions
What purpose does a CAPSULE serve?
Protection from phagocytosis
What purpose does a FLAGELLA serve?
Name two reasons we prepare Streak Plates
- 1. The isolation of pure colonies of bacteria from a sample containing a mixture of bacterial species.
- 2. To observe the morphology of the isolated colonies which aids in their classification.
What are the purposes of a Spread Plate?
- 1. To create a bacterial lawn.
- 2. To spread out a diluted sample of bacteria so that individual colonies can be seen.
Which experiments used the Spread Plate?
- Antibiotic and Disinfectant Testing
- Viral Plaque Assay
- Viable Plate Count
- Bacterial Transformations
What did the temperature experiment show?
The experiment examined 3 species of bacteria (P. fragi, B. stereothermophilus and S. aureus) to show which category they into on the basis of their preferred range of temperature.
What is a Psychrophile?
- Cold-Loving Microbes (-10°C to 20°C)
- P. fragi
What is a Thermophile?
- Heat-Loving Microbes (40°C to 70°C)
- B. stereothermphilus
Which enzyme does the Catalase Test detect?
- Detection of the enzyme Catalase.
- Catalase is possessed by some bacteria to convery the toxic byproduct Hydrogen Peroxide (H2O2) into water and oxygen (O2).
Which two organisms were used in the Catalase Test and what were the results?
and E. faecalis.
tested positive for Catalase by having effervescent bubbling after incubation, addition of 3% H2
tested negative for Catalase due to its lack of effervescent bubbling.
Which enzyme does the Oxidase Test detect?
- Detection of the cytochrome Oxidase.
- Oxidase is the final electron acceptor in the ETS and it's detection indicates ALL cytochromes in the ETS are present. (VERY active ETS - Aerobic Organisms).
Which four organisms were used in the Oxidase Test and what were the results?
E. faecalis, P. mirabilis, P. aeruginosa
and E. coli.
tested positive for Oxidase by turning blue when applied to the Oxidase DrySlide card.
E. faecalis, P. aeruginosa
and E. coli
all tested negative.
What enzyme does the Starch Hydrolysis Test detect?
- Detection of Amylase.
- Amylase is excreted from some bacteria in order to digest nutrients so they can absorb products for sources of energy. May environmental organisms digest starch so they can absorb the glucose for evergy production.
What two organisms were used in the Starch Hydroysis Test and what were the results?
and E. coli
testted positive for amylase (starch digestions) by the presence of a brown halo after incubation and iodine flooding.
What ezyme does the TSI Agar Test detect?
Determines which of 3 sugars (glucose, lactose or sucrose) organisms ferment and if they are capable of degrading Cysteine (a sulfur containing amino acid) to produce H2S (Hydrogen Sulfide).
Used to classify enteric (rod-shaped Gram negative) bacteria.
What three organisms were tested in the TSI Agar test and what were the results?
E. coli, P. mirabilis
and P. aeruginosa.
- Yellow throughout which indicated it is an ACID and fermented ALL 3 SUGARS.
- Red throughout which indicates it is alkaline and did not ferment any sugars.
- Black throughout which means it ferments glucose and produces H2
What are the patterns of growth in the agar deep for aerobes, anaerobes and facultative anaerobes?
- Agar Deep: Inculate TSA by stabbing agar completely to the butt of the tube.
- Aerobes will only grow where oxgen is present (top).
- Anaerobes will only grow where oxygen isn't present (butt).
- Falculatative Anaerobes will grow on the top, middle and bottom.
What are the patterns of growth in the Thioglycollate Medium for aerobes, anaerobes and facultative anaerobes?
Thioglycollate broth tubes have a red or pink ring around the top which creates an anaerobic environment.
- Aerobes will grow only at the top of the tube above the red ring.
- Anaerobes will grow underneath the red ring
- Falcultative Anaerobes will grow throughout the tube.
- Pictured: P. Aerug which is an aerobe.
What is the O2 indicator in Thioglycollate Medium?
Resazurin, which is red or pink in the presence of O2
What is the O2 indicator in the Anaerobe Jar?
Methylene Blue which is blue in the presence of O2
and colorless in its absence.
Of the organisms tested in the Thioglycollate Medium and Anaerobe Jar, which is a strict anaerobe and which is a strict aerobe?
- Strict Anaerobe: C. sporogenes
- Strict Aerobe: P. aeruginosa
How does the Anaerobe Jar work?
- The jar contains an envelope of chemicals, a catalyst (palladium) and an O2 indicator (methylene blue).
- The chemicals produce hydrogen and carbon dioxide which combine with oxygen to form water in the presence of palladium (the catalyst), thus removing oxygen from the atmosphere of the jar.
What enzyme was used in the experiment on Enzyme Kinetics?
HRPO - Horseradish peroxidase
What Substrates were used in the experiment on Enzyme Kinetics?
Hydrogen Peroxide and ABTS Solution
How are Serial Dilutions determined?
- By multiplying the number of colonies by the inverse of the dilution.
- Ex.: Count on plate 10-6 is 253. Serial dilution would be 2.53 x 108.
What two numbers are used to determine plate count?
How does UV light kill bacteria?
- UV light (which is non ionizing radiation) causes cell damage by making thymine dimers in DNA which interfere with DNA replication.
- The most effective germicidal wavelength is 260 nm.
What are the disadvantages of using UV light for disinfection?
- 1. Radiation is not very penetrating so the organisms to be killed must be directly exposed to the UV rays.
- 2. UV light can damage human eyes and prolonged use can cause burns and skin cancer in humans.
What are normal flora you would expect to see on the skin (hands)?
- Staphylococcus epidermidis
- Staphylococcus aureus
How were the inoculums used for Antibiotic and Disinfectant Testing standardized?
- Antibiotic: Using a Muellar-Hinton plate which has been previously inoculated with a standard amount of the microorganisms to be tested.
- Disinfectant: Adding enough og the organism to a sterile TSB broth tube until the turbidity matched No. 0.5 McFarland Standard.
How is Antibacterial activity measured?
By measuring the Zones of Inhibition
and placing the organism based on the Table of Sensitivity Standards
Of the organisms tested, which was Resistant to most Antibiotics?
Of the organisms tested, which was Susceptible to most Antibiotics?
Which Disinfectant worked best with each organism?
Which organism and media was used in the Membrane Filtration Experiment?
- E. coli
- EMB (Eosin Methylene Blue) plates
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