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144 Cards in this Set

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Test:

Glucose Fermentation
Durham Tube - Gas Production Test
Media:
Phenol Red Glucose Broth

Reactions:

Glucose ----------------> acids

Positive results:
Yellow media
Test:

Lactose Fermentation
Media:
Phenol Red Lactose Broth

Reactions:

Lactose ----------------> acids

Positive results:
Yellow media
Test:

Mannitol Fermentation
Media:
Phenol Red Mannitol Broth

Reactions:

Mannitol ----------------> acids

Positive results:
Yellow media
Test:

Mixed Acid Fermentation
Media:
MR - VP Broth

Reactions:

Glucose ----------------> acids

ADD: 4 drops of methyl red

Positive results:
Red media
Test:

Butanediol Fermentation
Media:
MR - VP Broth

Reactions:

2 Pyruvate --> Butanediol + Barritt's reagent

Positive results:
Pink to Red media
Test:

Starch Hydrolysis
Media:
TSI Slant

Reactions:

Starch ---------> maltose + glucose + Gram's iodine

Positive results:
CLEAR ZONE along streak
Test:

Catalase Production (Test)
Media:
TSA slant

Reactions:

2H O ----------> O + 2H O

Positive results:
Bubbles media
Test:

Urea Hydrolysis
Media:
Urea broth

Reactions:

Urea ----------> ammonia

Positive results:
Bright pink media
Test:

Tryptophan Hydrolysis (Degradation)
Media:
Tryptone broth

Reactions:

Tryptophan----------> Indole + Kovac's reagent

Positive results:
Red media
Test:

Citrate Utilization (Test)
Media:
Simmons Citrate Agar Slate

Reactions:

Citrate ----------> alkaline products

Positive results:
Prussian blue media
Test:

Nitrate Reduction
Media:
Nitrate broth

Reactions:

NO ----------> NO + Reagent A + Reagent B

Positive results:
Deep red media
Test:

Sugar Fermentation
Media:
TSI Slant

Reactions:

Glucose -------------> acids

Positive results:
Red slant/Yellow butt
Test:

Sugar Fermentation
Media:
TSI Slant

Reactions:

Glucose -------------> acids

Lactose -------------> acids

Sucrose -------------> acids

Positive results:
Yellow slant/Yellow butt
Test:

CO Production
Media:
TSI Slant

Reactions:

Gas Production

Positive results:
Bubbles/cracks
Test:

Hydrogen Sulfide Production
Media:
TSI Slant

Reactions:

Cysteine -------------> H S

Positive results:
Black butt
Test:

Casein Hydrolysis
Media:
Skim milk agar plate

Reactions:

Casein -----------> peptides + amino acids

Positive results:
CLEAR ZONE along streak
Aerobic
Requires oxygen
Agar
Complex polysaccharide derived from a marine alga (red) and used as a soldifying agent in culture media (agar by itself is NOT a nutrient)
Aseptic technique
To inoculate the sterile medium with a pure culture
of microorganisms without outside contamination.
Autoclave
A machine used to sterilize by the use of steam under pressure. Insures that the spores are destroyed. It will go up to fifteen pounds of pressure per square inch. It will be kept at 121 degrees centigrade for 15 to 20 minutes.
Broth
Medium without agar
Brownian Movement
Vibrations of an object seen in a microscope, not true motility.
CFU
Colony-forming units, i.e. colonies.
Colony
A macroscopically visible population of cells growing on solid medium, arising from a single cell.
Culture medium
Those nutrients essential for growth of a given microorganism.
Provides suitable surroundings for growth = the proper pH, osmotic pressure, oxygen, temperature, etc.
Differential Stain
Uses 2 or more dyes which allow differentiation between different bacterial groups or structures.
Counterstain
The 2nd dye added to a smear, taken in after the wall is decolorized.
Decolorizer
The reagent used to remove the primary dye from the cell wall in a differential stain, e.g. acid alcohol, acetone-alcohol.
Deep
Agar media that solidifies in a vertical tube.
EMB aqar
Eosin-methylene blue agar; contains bile salts and the dyes eosin and methylene blue, all inhibitory to Gram (+) bacteria, therefore, this medium selects for Gram (-) bacteria; it differentiates lactose-fermenting bacteria from non-lactose-fermenting bacteria.
Facultative anaerobe
Uses oxygen when present but can either ferment or anaerobically respire without it.
Fastidious
Hard-to-grow bacteria, requiring grow factors or particular nutrients.
Flaqella
A structure for motility.
Genus
Category of organisms with like features and closely related, divided into species.
Growth
The development of a population of cells from one or a few cells.
This is not growth in size
of the cells, but growth with respect to multiplication of the cells.
Inoculum
Material used to initiated a microbial culture.
Inoculate
The transfer of the cells you want to grow into a culture medium.
Halophilic
Salt-tolerant or salt-loving (salt-requiring).
Incubate
Cells placed in an environment providing suitable growth conditions.
MAC
MacConkey agar; contains bile salts and crystal violet, both inhibitory to Gram(+) bacteria, therefore, this medium selects for Gram(-) bacteria; it differentiates lactose-fermenting bacteria from non-lactose-fermenting bacteria.
MSA
Mannitol salt agar; contains 7.5% NaCI, which is inhibitory to most bacteria, therefore it selects for halophillic (halotolerant) bacteria; it differentiates mannitol-fermenting bacteria from non-mannitol
fermenting bacteria.
Mixed Culture
More than one species of microorganisms.
Obligate Aerobe
Requires oxygen to grow.
Obligate Anaerobe
Does not use oxygen to grow, may even be killed by it.
Pathogenic
Disease-causing.
Petri dish
Shallow dishes with a lid that nest over the bottom to prevent contamination.
Petri (or culture) Plate
Petri dish that is filled.
PFU
Plaque-forming units produced by bacterial viruses when infecting host bacterial cells.
Plaque
Destruction of the bacterial lawn by a bacteriophage
as the lytic infection progresses.
Pour Plate
Procedure where liquefied agar has been poured into a Petri dish after being mixed with bacteria.
Primary Stain
The 1st dye used in a differential stain,
e.g. malachite green,
crystal violet,
carbolfuchsin.
Pure culture
Single species of microorganisms.
Slant
Test tubes with agar media that are allowed to solidify with
the tube held at an angle
so that a large surface area for growth is available.
Species
A subdivision of a genus.
Sterile
Heated to a point where all organisms present
are destroyed. It may
not be heated, but basically the word sterile means absolutely no organisms.
Streak Plate
Procedure where a bacterial specimen is placed on a pre-made plate and diluted out using flame and multiple sections.
TSB
Trypticase soy broth
TSA
Trypticase soy agar
Turbidity
Cloudiness in liquid broth. The more bacteria,
the more turbidity there is.
Ubiquitous
Bacteria are universally distributed; they are everywhere.
Zone of Inhibition
Area of no bacterial growth around a chemical on a disc, indicates sensitivity.
Know the function of the streak plate technique
Streak plate for isolation
Type of media used for streak plate.
TSB
Understand the differences in cell wall structure of
gram(+) VS. gram(-)
bacteria
Gram(+):
THICK peptidoglycan
Teichoic acid

Gram (-):
THIN peptidoglycan
OUTER membrane
Periplasm
Be able to identify gram reaction under microscope or in diagram
Gram(+) Purple
Gram(-) Pink
Staphylococcus aureus
Gram(+), Coccus, Staphylococci

Habitats:
Lives on skin/in nose
Can cause skin infections, pneumoniae, Toxic Shock Syndrome
Escherichia coli
Gram Reaction:
Gram(-), Bacillus, Single bacillus

Habitats:
Lives in the lower intestines of mammals
Can cause food poisoning; UTIs
Micrococcus luteus
Gram(+), Coccus, Tetrads

Habitats:
Found in soil, dust, water, air and normal flora of the mammalian skin
Non-pathogenic but a contaminant
Serratia marcescens
Gram(-), Bacillus, Coccobacillus

Habitats:
Lives in damp conditions(bathroom, pink discoloration)
Pathogenic (involved in nosocomial infections)
UTI's
Enterococcus faecalis
Gram(+),Coccus,Streptococci

Habitats:
Inhabits the GI tracts of human and other mammals
Can cause infections in humans especially in nosocomial (hospital) environment
Pseudomonas aeruginosa
Gram(-),Bacillus, Single bacillus

Habitats:
Common inhabitants of soil & water; occur regularly on surfaces of plants and occasionally on the surfaces of animals
Bacillus subtilis
Gram(+),Bacillus,Single bacillus

Habitats:
Able to form tough, protective endospores wihich allows organism to tolerate extreme environmental conditions
Not considered human pathogens
MAY contaminate foods
Be able to determine CPUs
Organisms/ml =
# colonies X inverse of dilution

1: 100,000

80 colonies X 100,000 = 8,000,000
8.0 x 10 CFU/ml
Antimicrobic Sensitivty Test

What method was used?
Kirby-Bauer Method
What type of AGAR?
Mueller-Hinton II agar
What antiobiotics were used in lab?
Tetracycline
Ampicillin
Pencillin
Sulfamethoxazole
Chloramphenicol
Tobramycin
What are the modes of actions of antibiotics?

Tetracycline
Inhibits protein synthesis
What are the modes of actions of antibiotics?

Ampicillin
Inhibits cell wall synthesis
What are the modes of actions of antibiotics?

Pencillin
Inhibits cell wall synthesis
What are the modes of actions of antibiotics?

Sulfamethoxazole
Inhibits nucleic acid synthesis
What are the modes of actions of antibiotics?

Chloramphenicol
Inhibits protein synthesis
What are the modes of actions of antibiotics?

Tobramycin
Inhibits protein synthesis
How did our species respond to the six different antibiotics?

Staphylococcus aureus
Sensitive to all 6 antibiotics.
How did our species respond to the six different antibiotics?

Escherichia coli
RESISTANT to Penicillin; sensitive to Ampicillin, Sulfamethoxazole, Tetracycline, Tobramycin, and Chloramphenicol.
How did our species respond to the six different antibiotics?

Pseudomonas aeruginosa
RESISTANT to Penicillin, Ampicillin, Sulfamethoxazole, & Chloramphenicol; INTERMEDIATE to Tetracycline; sensitive to Tobramycin.
What is an antiseptic?
Chemicals applied to animal surfaces (skin/mucous membrane)to prevent infection by killing or inhibiting the growth of pathogens
What antiseptics were tested?
Iodine
Alcohol
Listerine
ANTISEPTIC

What type of agar was used?
TSA
How did our species respond to the three different antiseptics?

Staphylococcus aureus

Most effective?
Greatest to least:
Iodine, Alcohol, Listerine.

I A L

MOST EFFECTIVE: Iodine
How did our species respond to the three different antiseptics?

Pseudomonas aeruginosa
Most effective?
Greatest to least:
Alcohol, Iodine, Listerine.

A I L

Most effective: ALCOHOL
How did our species respond to the three different antiseptics?

Escherichia coli
Most effective?
Greatest to least:
Alcohol, Iodine, Listerine.

A I L

Most effective: ALCOHOL
What is a disinfectant?
Agents, usually chemicals, used to disinfect inanimate objects
DISINFECTANT

What species of bacteria were tested?
Escherichia coli
Bacillus subtilus
DISINFECTANT

Compare and contrast the two species tested.

What were their growth patterns like and why?
Bacillus subtilis (gram(+)) was more resistant to disinfectants than Escherichia coli because of its ability to form endospores in the harsh chemical environment and Gram(-).
What was the MOST EFFECTIVE disinfectant against each species?

Escherichia coli
PINE SOL, Lysol, Bleach
What was the MOST EFFECTIVE disinfectant against each species?

Bacillus subtilis
LYSOL, Bleach, Pine sol

(Effectiveness was the same at 1 min, 5 min, & 10 min)
What disinfectants were tested?
Lysol
Pine sol
Bleach
What is a phage?
Virus
Be able to determine PFUs (organism/ml = # of plaques x inverse of dilution)
Plate 1:
Phage particles/mL = # of plaques X dilution factor

Phage particles/mL = 500 X 1,000,000
Phage particles/mL = 500,000,000 -> 5.0 X 10 PFU/mL
Be able to determine PFUs (organism/ml = # of plaques x inverse of dilution)
Plate 2:
Phage particles/mL = # of plaques X dilution factor

Phage particles/mL = 50 X 10,000,000
Phage particles/mL = 500,000,000 -> 5.0 X 10 PFU/mL
Be able to determine PFUs (organism/ml = # of plaques x inverse of dilution)
Plate 3:
Phage particles/mL = # of plaques X dilution factor

Phage particles/mL - 5 X 100,000,000
Phage particles/mL - 500,000,000 -> 5.0 X 10 PFU/mL

Phage particles/mL - 500,000,000 -> 5.0 X 10 PFU/mL

*In this example, the bacteriophage titer (number of phage particles
in the original stock
of 74) is 5.0 X 108 PFU/mL.
Differential Stain:

Primary stain
Mordant
Decolorizer
Counterstain
Gram Stain:

Crystal Violet
Gram’s Iodine
Ethanol
Safranin
Microaerophilic
Likes a reduced oxygen concentration.
Shapes:

Coccus (cocci)
Bacillus (bacilli)
Spiral (curve rods)
Shapes
What colony characteristics can be used for differentiation of bacterial species? As an example, compare the properties of colonies of Serratia marcescens and Micrococcus luteus on your streak plate. (EX. 9)
Color, shape of colonies
Coccus Arrangements:

Single coccus
Diplococci
Tetrads
Sarcinae
Streptococci
Staphylococci
`
Staphylococcus epidermis
Gram(+),Cocci,Staphylococci

Habitats:
Occurs frequently on the skin of humans & animals and in mucous membranes
Myobacterium smegmatis
NOT truly gram(+) NOR gram (-)
Bacillus, Single bacillus

Habitats:
Found in normal genital secretions
Proteus mirabilis
Gram(-), Bacillus, Single bacillus

Habitats:
Cause UTIs, wound infections, septicemia & pneumonias
Habitat is human intestinal tract, soil, water & plants
Klebsiella pneumoniae
Gram(-), Bacillus, Single bacillus

Habitats:
Can cause pneumonia due to capsule
Found in the normal flora of mouth, skin & intestines
T4 Figure

Know Structure
Structure
What is a bacteriphage?
Viruses that infect bacteria
Species specificity of T4
E. Coli, Strain B
Pseudomonas aeruginosa after primary stain is added.(EX.14)
Purple
Bacterial colonies (CFU)
`
Test:

Antibiotic Sensitivity Testing
Media:

Mueller Hinton Agar plate
Test:

Antiseptic Sensitivity Testing
Media:

Trypticase Soy Agar plate
Test:

Disinfectant Sensitivity Testing
Media:

Trypticase Soy Broth
Test:

Gram Stain
Media:

Trypticase Soy Agar plate
Spiral Arrangments:

Vibrio
Spirillum
Spirochete
Arrangements
Test:

Bacteriophage Titer determination
Media:

Soft Agar/TSA plate
In regards to bacterial growth on solid media, define the term "colony".(EX. 9)
A macroscopically visible population of cells growing on solid medium, rising from a single cell.
Why is the loop flamed before it is placed in a culture tube? Why is it flamed after completing the inoc¬ulation?(EX. 9)
To sterilize the loop for aseptic technique
Provide two reasons why plates should be inverted during incubation.(EX. 9)
1.The problem of moisture on the cover lid is eliminated.
2. Moisture can become deposited on the agar surface causing the organisms to spread over the surface (defeating the isolation process.)
How does smear preparation of cells from a liquid medium differ from preparation of cells from a solid medium? (EX.10)
You must add a drop of water to suspend the organism in when obtaining it from
a solid medium
Why is it important to limit the quantity of cells used
to prepare a smear?
(EX.10)
To see individual cells
For preparation of a smear on a slide, what is the purpose of heat fixation? What problems can arise when the slide is heated in a flame? (EX.10)
1.To adhere to a slide
2.To kill m.o. destroy/denature part or all of the cell microorganisms
Which of the three differential stains would likely be the first
used when identifying an unknown bacterium? Explain. (EX.14)
Gram stain – it tells you if it is gram (+) or (-) and tells you the shape/arrangement of cells.
What is the function of a mordant? (EX.14)
(To adhere stain) Form a crystal complex with crystal violet so that
gram(+) bacteria retain the purple stain.
For differential staining, how does a counterstain differ from
a primary stain? (EX.14)
It is a less intense stain
How do gram-positive and gram-negative bacteria differ in cellular structure? How does this contribute to their differential staining properties? (EX.14)
Cell walls differ:
gram (+) - thick layer of peptidoglycan and teichoic acids
gram (-) – thin layer of peptidoglycan, periplasm, outer membrane
Which is the most critical step in the Gram-stain procedure? Why?
If this procedure is
done incorrectly, how
might that affect the
final results? (EX.14)
Decolorizing with ethanol; if left too long it can discolor the gram (+) cells
How does culture age affect the results of a Gram-stain? (EX.14)
Grams (+) can convert to gram-variable or gram (-)
Why must smear thickness be considered before
performing a Gram-stain? (EX.14)
If a smear is too thick you will not see individual cells
Bacteriophage plaques(PFU)
Plaques are clear zones formed in a lawn of cells due to lysis
by phage.
What color are bacterial endospores after a
Gram-stain is performed?

What does this tell you
about the physical properties of endospores?(EX.14)
Colorless

Endospores are highly resistant to stains, heat, etc…
Staphylococcus aureus before primary stain is added.(EX.14)
No color
Bacillus megaterium after the mordant is added.(EX.14)
Purple
Staphylococcus aureus cells after the decolorizer is used. (EX.14)
Purple
Moraxella (Branhamella) catarrhalis after the decolorizer is used. (EX.14)
No color-gram(-)
Bacillus megaterium after the counterstain is added. (EX.14)
Purple
Bacillus Arrangements:

Single Bacillus
Coccobacillus
Diplobacilli
Streptobacillus
`
Pseudomonas aeruginosa after the counterstain is added.(EX.14)
Pink
Why has the classification of slime molds been difficult? (Ex. 20)
`