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Define Aseptic technique.
refers to a procedure that is performed under sterile conditions
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A liquid that is dense and cloudy is known as ___.
Turbid
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A ____ ____ is one that contains only a single species of organism
Pure culture
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Separate masses of bacteria growing on a medium are known as a ___.
Colony
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Define streak plate technique.
Streaking is a technique used to isolate a pure strain from a single species of microorganism
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A ____ consists of one or more species of microorganisms growing in a nutrient growth medium often at specific temp
Culture
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A culture consisting of ONLY 1 species of microorganism is called a what?
Pure culture
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The ___ ___ technique permits one to see unstained bacteria on a stained background & determine their morphology.
Negative stain
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What stain do we use as the primary stain for spore stain?
Malachite Green
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What indicator do we use to test for starch digestion and how does it indicate?
- Iodine
- It is added to determine whether starch is still present or has been digested
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What does starch normally do in the presence of iodine?
Forms a blue black chemical complex
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Certain bacteria produce the enzyme ____ which digests DNA into its constituent nucleotides.
DNase
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How do we know if catalase is being produced by a bacteria?
If oxygen bubbles appear & the area effervesces w/the application of 3% hydrogen peroxide
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The enzyme ___ ___ breaks down the amino acid cysteine to form ___ & ___ ___.
- Cysteine desulfurase
- Alanine & hydrogen sulfide
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What term is used to describe when an outside microbe fr the floor, air, counter etc gets into your subculture?
Contamination
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____is the placement of something that will grow or reproduce into a medium.
Inoculation
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___ ___ is defined as the ability of a microscope to distinguish two close together images as being separate
Resolving power
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What is the name given to a substance that has the same refractive index as glass and keeps light in a straight line as it passes fr the slide to the objective lense?
Immersion oil
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What does parfocal mean?
An objective in view under one objective will still be in view under the other lenses
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Explain simple stain.
Simple stain is a positively charged basic stain that is attracted to negatively charged cells. The cells are stained and show against a clear background
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Explain Gram staining.
Stain technique that uses crystal violet, iodine, alcohol & safranin. Gram pos retain crystal violet & stain blue, Gram neg don't retain crystal violet & stain red fr safranin.
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What is it that makes Gram pos cell walls retain the crystal violet/iodine complex and causes Gram neg walls not too?
Gram pos bacteria have larger amts of peptidoglycan in their cell walls that traps the complex whereas Gram neg walls have less & fail to retain it
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Nigrosin, congo red & India ink are ___ stains that carry a ___ charge and are therefore repelled by ____ charged bacteria.
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What are 2 advantages to the negative stain technique?
- It does not distort bacterial cells
- Some cells such as spirochetes dont stain well w/other techniques
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What is aseptic transfer?
Sterile, microbe free mvmt or transfer of microorganisms fr one medium to another
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When Gram staining bacteria that retain crystal violet stain are known as ___-___ & those that do not are ___-___.
- Gram positive
- Gram negative
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___ ___ & ____ form a complex that exits easily fr Gram neg bacteria but not fr Gram pos when ___ is applied.
- Crystal violet
- Iodine
- Alcohol
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Endospores are formed by members of several gram-___ bacterial genera such as ____ &____.
- Positive
- Bacillus and clostridium
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What are 3 characteristics of endospores?
- Resist boiling for 2 hrs or more
- Contain little water
- Exhibit few chem reactions
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The iodine in a Gram stain acts as a mordant which means what?
It increases the affinity of dye to the cell by forming a complex w/the stain
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Crystal violet is the ___ ___ when performing the Gram stain tchenique
Primary stain
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What is the counter stain when performing the gram stain technique
Safranin
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What is the purpose of the spore stain technique?
To contrast vegetative cells fr endospores
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Why is it that bacterial endospores cannot be penetrated easily by stain using simple or Gram stain techniques?
B/c they contain numerous protective layers
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What do we do to assist with stain penetration when staining bacterial endospores?
Apply heat
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What stain do we use as the primary stain for spore stain?
Malachite green
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When spore staining we counterstain w/___ for __ min. This will have what effect?
- Safranin for 1 min
- This will stain the vegetative cells but have no effect on the spores
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Explain the hanging drop test?
Used to test for motility, a 2-3 loopfuls of bacterial broth culture are placed on a coverslip w/petroleum jelly on all 4 corners. An inverted depression slide is placed over the coverslip & the whole thing is inverted to be viewed
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What are 2 methods for determining motility?
- Hanging drop technique
- Utilizing motility agar
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What characteristics of motility should we note when viewing our slide?
- Patterns of motion
- Where cells accumulate
- Relative shapes & sizes
- Configurations displayed
- Speed of mvmt
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Define spore stain.
A 2 part staining technique that uses malachite green & safranin to create contrast between vegetative cells & endospores. Vegetative cells will be red/orange & spores will be green
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What are the 3 spore positions w/regard to vegetative cells?
Central, Subterminal, Terminal
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What is phenol red & what is it used for?
An indicator for pH that is red when in neutral or basic solutions & bright yellow in the presence of acid
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What is thioglycollate media?
- Thioglycollate is media used to test for oxygen requirements of a bacteria.
- Growth @ top = aerobic
- Growth @ bottom = anaerobic
- Growth throughout = facultative
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What do biochemical tests tell us?
- Serve as fingerprints for bacterial Results
- Combined results help us to ID organisms & diagnose a disease
- Show role played by organisms in the environment & how organic substances are degraded & products formed
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Std biological test results for organisms can be found where?
Bergey's Manual of Systematic Bacteriology
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____ is a type of microbial metabolism in which an organic intermediary molecule serves as electron acceptor.
Fermentation
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What is a Durham tube?
A sm vial inverted in a test tube testing for carb fermentation to collect gas
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A positive test for carb fermentation is indicated by what?
The production of acid & gas in the tube b/c these are byproducts of fermentation
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___ is a polysaccharide consisting of thousands of glucose molecules chemically bonded to one another
Starch
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What is the enzyme amylase used for?
Enzyme used to biochemically break bonds between glucose monomers
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How do we test for starch digestion?
By inoculating bacteria to starch agar medium, incubating, then adding iodine to determine whether starch is still present
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What determines a positive result for starch digestion?
Digestion has occurred if areas around the bacterial streak remain clear or lt brown due to iodine color. Areas that turn blue black are showing the presence of starch in the agar
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What is catalase?
An enzyme that breaks down hydrogen peroxide into oxygen & water b/f it accumulates & damages cells
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How do we test for catalase?
Place several drops do 3% H2O2on bacterial streak. A pos result is indicated by bubbling & effervescing @ the site indicating H2O2 is being broken down
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The enzyme ___ digests DNA into its constituent nucleotides.
DNase
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How do we test for the presence of DNase & what constitutes a pos result?
Incubate bacteria on med containing DNA then add HCL aft incubation. If area around streak remains clear & rest of plate turns cloudy the test is pos for digestion
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Certain bacteria have the ability to produce an enzyme called ___ ___ that breaks down cysteine into ___ & ___ ___.
- Cysteine desulfurase
- Alanine & hydrogen sulfide
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What medium do we use to test for hydrogen sulfide production & why?
Sulfide Indole Motility (SIM) medium b/c it contains cysteine & iron ions
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What three things can be tested w/ SIM medium?
- Hydrogen sulfide production
- Indole production
- Motility
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What constitutes a pos test for the production of hydrogen sulfide? What causes a pos result?
Blackening along line of inoculation & possibly the entire tube caused by hydrogen sulfide reacting w/Fe ions in the agar
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The end product of protein metabolism in bacteria is ___ which can be broken down by the enzyme ___ into ___ & ___ ___.
- Urea
- Urease
- Ammonia & carbon dioxide
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How do we test for urea digestion & what indicates a pos result?
- Inoculate nutrient broth containing phenol red indicator.
- If urea digested (+) during incubation, ammonia will raise pH causing indicator to become deep fuchsia or purple
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Some bacteria contain lipases which do what?
Hydrolyze linkages in lipids separating glycerol molecule fr it's fatty acid molecules
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Lipid digesting bacteria cause a type of food spoilage in which foul odors and flavors develop b/c of fatty acids released fr lipids.
Rancidity
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What are some characteristics of spirit blue agar?
- Rich in lipids
- pH indicator called spirit blue
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What happens to spirit blue agar in the presence of acids & bases?
- Acid -- turns deep blue
- neutral -- pale lavender
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What indicates a pos result for lipid digestion?
Spirit blue agar medium will become deep blue as lipids are digested, fatty acids accumulate and pH lowers
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What is Casein?
Milk protein
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What are proteinases?
Enzymes produced by bacteria that allow it to digest casein
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How did we test for casein digestion?
- We inoculated a tube filled with Litmus Milk.
- A pos result shows pink on top indicating acid production & a solid curd in the tube
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Which bacteria that we studied are Gram Neg?
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Of the bacteria we have worked with which are Gram Pos?
- M. luteus
- B. cereus
- E. faecalis
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E. coli produced both acid and gas indicating it fermented which of the carbohydrates we tested?
- Glucose
- Lactose
- Fructose
- Maltose
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E. coli, M. luteus, P. fluorescens, E. faecalis are ___ for starch digestion
negative
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B. cereus is ___ fr starch digestion
positive
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Which of these bacteria are positive for catalase production. . E. coli, M. luteus, B. cereus, E. faecalis, P. fluorescens
- E. coli
- M. luteus
- B. cereus
- P. fluorescens
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Which of the following are positive for DNA digestion?
E. coli
M. luteus
B. cereus
E. faecalis
P. fluorescens
- B. cereus
- All the rest are Negative
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Which of the following are positive for Hydrogen Sulfide Production?
E. coli
M. luteus
B. cereus
E. faecalis
P. fluorescens
None
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Which of the following are positive for Urea Digestion?
E. coli
M. luteus
B. cereus
E. faecalis
P. fluorescens
P. fluorescens is positive & the rest are negative
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Which of the following are positive for lipid digestion?
E. coli
M. luteus
B. cereus
E. faecalis
P. fluorescens
E. faecalis & M. luteus are positive for lipid digestion while the others are not
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Which of the following are positive for casein digestion?
E. coli
P. fluorescens
P. fluorescens is pos for casein digestion while E. coli is negative
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Which of the following bacteria produce DNase
E. coli
M. luteus
B. cereus
E. faecalis
P. fluorescens
B. cereus
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What is tryptic soy agar?
general purpose medium , providing enough nutrients to allow for a wide variety of microorganisms to grow
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What is tryptic soy broth?
culture broth to grow aerobic bacteria
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Describe Spirit Blue Agar.
Spirit blue agar is a lipid rich nutrient medium with the dye spirit blue present. It is used to test for lipid digestion by lipases
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Describe DNase agar.
DNase agar contains nutrients for the bacteria, DNA, and methyl green as an indicator.
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What do fermentation tubes contain?
Phenol red to monitor pH & a Durham tube to determine if gas is produced
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What is contained on a "Urea test" test tube?
Nutrient broth that contains urea & phenol red
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Which of the following bacteria are spore formers?
E. coli
M. luteus
B. cereus
E. faecalis
P. fluorescens
B. cereus only
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