LAB EXAM #1 experiments deck Flashcards
Aseptic technique:
Insures that there is no contamination of microorganisms
Loops are used to:
Transfer from liquid media to liquid media or petri plates
Needles are used to:
Transfer from solid media to solid media or petri plates
Purpose of smear prep:
To prepare the organism for staining
Smear preps are used to determine:
- Cell shape
- Arrangement of cells
- Internal structures
Procedure of aseptic technique:
- Transfer org to slant
- Transfer org from slant to broth
- Transfer org from broth to plate
Procedure of smear prep from a broth: (3)
- Using AT, transfer 3 loops of org to the middle of the slide
- Air dry
- Heat-fix slide by passing slide through the bunsen burner flame 5 times
Procedure of smear prep from a solid media: (4)
- Using AT, place 3 loops of WATER onto center of slide
- Transfer org to the water
- Air dry
- Heat-fix the slide by passing through the bunsen burner 5 times
Purpose of heat-fixation of smear preps?
Kills the organism and adheres them to the slide so they aren’t washed off during staining
Purpose of simple staining procedure: (2)
- Used to make bacterial cells easier to visualize on the slide
- Used to determine bacterial cell morphology (cell shape)
2 types of dyes for staining:
Basic dye
Acidic dye
Basic dye:
- Positively charged chromophore
- Works well with bacterial cells because they have a negative charge
- Stains inside of cell
Acidic dye:
- Negatively charged chromophore
- Repelled by the negative charge of the bacterial cell
- Stains outside of cell
3 main shapes of bacteria:
- Rods (bacilli)
- Cocci (spherical)
- Spiral or curved
Dye use in simple staining experiment:
Methylene blue
Procedure for simple staining: (4)
- Using AT, make a smear prep by adding org to middle of slide
- Air dry and then heat-fix slide
- Add methylene blue
- Rinse slide and view under oil
Purpose of negative stain:
To demonstrate the size and shape of bacteria
Appearance of bacterial cell in a negative stain:
Negative stain stains the background of bacterial cell and bacterial cell will appear transparent against a dark background
Dye used in negative stain experiment?
Nigrosin or india ink
Procedure for negative stain experiment: (4)
- Add 1 drop of india ink near end of slide.
- Transfer org into india ink drop
- With another slide, place the edge of the new slide against the drop of india ink and push the spreader slide across the slide
- Air dry then view under oil
2 types of gel-like layer on the outside of the cell wall:
- Capsule layer
2. Slime layer
Capsule layer: (3)
- Gelatinous layer
- Attached tightly to bacteria
- Made of polysaccharides known as glycocalyx
Slime layer:
- Layer made of ECM
- Loosely attached to bacteria
Function of capsule and slime layer:
- Help cells from being engulfed or destroyed
- Attachment to solid surfaces
In the capsule staining experiment, what is the first step?
Negative staining procedure
Under oil, how do capsule appear?
Halos around pink/red cells with a dark background
Dyes used in capsule staining experiment:
Include charges
- Congo red: acidic dye, stains background
- Maneval’s: basic dye, stains inside of cell
Procedure of capsule staining experiment: (7)
- Add 1 drop of congo red to end of slide
- Transfer org into congo red
- With another slide, place the edge of the new slide against the drop of congo red and push the spreader slide across the slide
- Air dry
- Add Maneval’s, as the counterstain, to the congo red
- Gently rinse
- Air dry and view under oIL
Hans Christian Gram:
Danish physician that developed a staining technique to differentiate bacterial cells from eukaryotic nuclei
Purpose of gram stain:
Provides identification of unknown bacteria
Gram positive cells:
- Appear purple
- 90% peptidoglycan 10% teichoic acids
Gram negative cells:
- Appear pink or red
- 10% peptidoglycan 90% outer membrane
Purpose of crystal violet in gram stain?
Primary stain that gives it the purple color
Purpose of gram’s iodine in gram stain?
Mordant- combines insoluble complex with crystal violet in gram positive cells
Purpose of 95% ethanol in gram stain?
Decolorizing agent- removes purple from gram negative cells
Purpose of safranin in gram stain?
Counterstain for gram negative cells so they may be seen as pink or red
Dyes used in gram stain experiment: (4)
- crystal violet
- gram’s iodine
- 95% ethanol
- safranin
Procedure for gram stain experiment? (6)
- Make a smear prep by adding org to middle of slide
- Heat-fix
- Cover with crystal violet then rinse
- Cover with gram’s iodine then pour off
- Decolorize with 95% ethanol then rinse
- Counterstain with safranin then rinse and view under oil
Purpose of acid-fast staining:
Used to identify Mycobacterium and Nocardia that have mycolic acid in cell wall
Acid-fast bacteria are stained:
Pink to red by the dye basic fuchsin
Non-acid fast bacteria are stained:
Blue with the counterstain methylene blue
Dyes used in the acid-fast staining experiment: (3)
- Carbolfuchsin
- Acid alcohol
- Methylene blue (counterstain)
Procedure for acid-fast staining experiment: (5)
- Make a smear prep by adding org to middle of slide
- Cover with carbolfuchsin for 5 min
- Gently rinse
- Decolorize with acid alcohol then rinse
- Counterstain with methylene blue then rinse and view under oil
2 species of bacteria that form endospores are:
Bacillus and Clostridia
How to enter an endospore in endospore staining?
Heat needs to be applied to help the stain enter the endospore- mordant
In the endospore staining experiment, what color will the endospore and vegetative cell be?
Endospore: green due to malachite green
Vegetative cell: red due to safranin
Dyes used in the endospore staining experiment:
Malachite green
Safranin-counter stain
Procedure for endospore staining experiment:
- Make a smear prep by adding org to middle of slide
- Cover with malachite green
- Heat-fix and replenish malachite green as needed
- Rinse
- Counterstain with safranin then rinse and view under oil
Motility is found in:
Spirochetes and rod shaped bacteria
Purpose of motility experiment?
To identify the presence of flagella
Procedure for wet mount in motility experiment:
- Make a smear prep by adding org to middle of slide
- Cover slide with cover slip
- View under 400x immediately
Procedure for motility agar in motility experiment:
- Take one tube with TTC in it and stab needle into agar for org
- Incubate media at 25C
What is TTC?
A colorless dye that when bacteria reduce it, it becomes red
Motile organisms:
Move away from stab line and have a fuzzy appearance
Motile organisms that can reduce the TTC:
Move away from stab line and have red fuzzy appearance
Non-motile organisms:
Will not move away from stab line and the rest of the media will be clear
Non-motile organisms that can reduce the TTC:
Red color in the stab line only and the rest will b e clear
Who was the first person to utilize the pure culture technique?
Robert Koch
Pure culture technique:
Helps obtain a single kind of organism from a mixed culture
2 commonly use methods of the pure culture technique:
- Pour plate
2. Streak plate
Streak plate method for the pure culture technique experiment:
Streak TSA plate with org using a quadrant streak
Pour plate method for pure culture technique experiment: (6)
Day 1:
- Obtain 3 petri plates
- Obtain 3 TSA tubes from the water bath and transfer the mixed culture into tube #1, mix
- Before pouring into petri plate, transfer 1 loop from tube #1 into tube #2
- Pour tube #1 into petri plate
- Transfer 1 loop from tube #2 into tube #3, then pour tube #2 into petri plate
- Pour tube #3 into petri plate
- Gently rotate plate to mix content then flip upside down when agar has solidified
Day 2:
- Obtain 3 TSA slants and transfer 1 colony of each color onto each slant
- Incubate at 25C
UV light falls between:
4nm and 400 nm
Purpose of UV light effects experiment:
To demonstrate bacteriostatic effect
3 ways that UV light damages our cells:
UVA: tanning beds
UVB: damaging cancer
UVC: germicidal lamps, hoods not seen in nature-> absorbed by O2
Procedure for UV light effects experiment:
- Obtain TSA plate and sterile swab
- Swab plate with organism
- When it is your exposure time, place plate in biosafety cabinet with a flash card covering half of the plate
- Flip over and examine number of colonies exposed to the UV light next lab period
Enumeraton:
Number of viable and non-viable microbes in a sample
Purpose of enumeration of bacteria experiment:
Determine the concentration of an organism
How is turbidity measured?
With a spectrophotometer where % transmittance or absorbance is obtained
Why is turbidity considered an indirect method?
It uses absorption of light to determine the amount of colloidal material in suspension
E. coli charge and shape:
Gram (-) rod
B. cereus: charge and shape:
Gram (+) rod
S. aureus charge and shape:
Gram (+) cocci
Primary lethal effect of UV light:
At 260nm, DNA absorbs the UV light and bonds are broken at its maximum-> pyrimidine dimers formed
Pyrimidine dimers:
Covalent bonds that form 2 adjacent thiamine or cytosine molecules of DNA-> changes shape of DNA-> can’t replicate
Purpose of effects of temp on growth exp:
To see the effects of temp on growth of organisms
Procedure for effects of temp on growth experiment:
Day 1
- Obtain 6 TSA tubes and label different temps
- Inoculate each tube with org
- Obtain 2 TSA slants and label different temps
- Inoculate with 2nd org
Day 2
- Measure %transmittance of each temp tube
- Determine OD and pigment production for each temp tube
Procedure for antiseptics mouth experiment:
Day 1
- Swab inside of mouth and place swab into TSA tube
- Incubate
Day 2
- Obtain TSA plate and divide into 3 quadrants
- Label each with scope, listerine, and cepacol
- Streak plate with mouth swab
- Place disks into each quadrant
Procedure for antiseptics assigned org experiment:
- Obtain TSA plate and divide into 3 quadrants
- Label each with: 70% isopropanol, bactine, and povidone iodine
- Streak plate with assigned org
- Place disks into each quadrant
T or F
E. coli is apart of Enterobacteriaceoe
T
T or F
E. faecalis is apart of streptococci as well as an enterococci:
T
Most effective antiseptic:
Least effective antiseptic:
Scope
Cepacol
Most susceptible org:
Least susceptible org:
Bactine
70% isopropanol
Procedure for antibiotic sensitivity experiment: Kirby-Bauer Method
Day 1
- Obtain a Mueller-Hinton plate (big plate)
- Swab plate with org
- Place abx disc on plate
Day 2
1. Measure zone of inhibition for each abx disc on plate
Indirect methods of enumeration of bacteria experiment:
Turbidity
Direct methods of enumeration of bacteria experiment:
- bacterial count
- standard plate count
- most probable number
Why is turbidity not a good test?
Contribution of dead and living cells
Formula for OD:
OD= 2-log(%T)
What is the difference between the methods?
Turbidity determines if sample has bacteria in it
Direct methods are a bacterial count
Purpose of slant tube:
More surface area
