Lab Test 1 Flashcards
What is the Kinyoun Stain used for
Acid-fast species of the bacterial genus Mycobacterium. It is a variation of a method developed by Robert Koch in 1882
Which was the Mordant used in the Acid-fast stain
DMSO
In our lab what experiment did we use: M. Smegmatis & S. epidermidis
What were the results
Describe appearance of each
Acid-fast stain
Staph epidermis = Not acid fast (Cocci Blue)
Mycobacterium smegmatis = Acid fast. (Pink Bacillus)
Why is DMSO used in fast-acid staining?
Which stain does it allow to penetrante & attach to the Waxy Cell Wall?
It is a Surfacent and allows Carbol Fuchsin to penetrante the Waxy cell wall
Carbol Fuchsin
Which type of bacteria are associated with Acid-fast cells
Why?
Mycobacteria
They have a Waxy Cell Wall
A Positive indication for Acid-Fast bacteria is….
A negative is
Positive = Red / Pink
Negative = Blue
Name the alcohol used in Acid Fast staining
Name the alcohol used in gram staining
Acid fast = Acid Alcohol
Gram staining = Aceton Alcohol
2 stains used in Acid Fast in Order
Are they basic or acidic
Positive or Negative charged
Reagent
Decolorizer
Carbol Fuchsin
Methylene Blue
Basic
DSMO
Acid Alcohol
Positive charged
How long does the DMSO-carbol fuchsin stay on the slide
4 minutes
How many passes with the Acid Alcohol in the Acid-Fast decolorizing technique
How many passes with the Aceton Alcohol in the Gram-staining Tech
1 pass
2 passes
The fast Acid cells are described as “pleomorphic”
True or False
True
When do you blot slides?
At the very end
Name the reagents used for gram-staining in order.
How long do you leave each on for?
- Crystal Violet 1 min (Primary Stain)
- Grams (Lugols) Iodine 1 minute (Mordant)
- Acetone alcohol (Decolorizer) Remove instantly
- Safranin 1 minute (Counter Stain)
Decolorizer, decoloizes….
Gram-negative & non acid fast
In the Gram Staining experiment which 2 bacteria were looked at?
What were the results?
Staphylococcus epidermidis = Gram Positive (Purple Grapes)
Escherichia coli = Gram Negative (Red Rods)
Name of paper for drying slide
Bibulous paper
Wet mount 4 steps
- Loopful of culture
- 1 drop of stain
- Cover slip
- View with 4, 10, 40x
NO Oil Immersion
Difference between Iris Diaphragm & Condensor lens
Iris Allows more light
Condenser Focuses it
What does parfocal mean
Ability to switch magnification without readjusting
2 physical characteristics of Corynebacterium
Rod shapped bacillus
Palisades arrangements
Gram-positive
Staphylococcus is gram-negative or gram-positive?
Describe shape and arrangement/ color after gram stain
Gram-positive
Round clusters like grapes
Purple
What are chromophores
Color part of stain
Difference between basic and acidic dyes
Basic dyes have a positive charge and adhere to the cell (due to the cell having a negative charge. )
Acidic dyes have a negative charge and stain the background (allowing the cell to be seen)
Crystal Violet is an example of what kind of stain?
Simple stain
Where are fusiform bacteria found in humans
Mouth
Can you ID bacterial species using a Simple Stained slide?
No, simple stain only IDs the morphology
How do we store and label petri dishes?
Why?
Label and store “upside-down”
To prevent condensation
Disinfectants are effective against which type of pathogens?
Which are they not effective against?
Vegetative cells & viruses
Not effective against ENDOSPORES
Which is our disinfectant in the laboratory?
Vesphene
This type of medium allows for growing of isolated colonies and are easy to observe
Disadvantage: they can dry out easily
Agar plates
These allow for growing large number of bacteria and at varying oxygen levels
Disadvantage: you cannot isolate them
Broths
These dry out less easily than agar palates and take up less space.
But have less visibility than agar plates
Slants
Retrieving a culture from a tube with a loop, the cap of the tube is removed and held with the fingers from which hand?
The one with the loop
Kinyoun Method is for what
Acid fast / Mycobacterium
The Schaeffer-Fulton method is also called…
(Endo)Spore Stain
How long do we let the Methylene Blue stay on the Kinyoun Method?
30 seconds
The endospore species is…
Where is it found? Aerobic ?
Gram-negative or gram-positive
Morphology and arrangements
Bacillus Cereus
Soil/ Yes
Gram-positive
Streptobacillis
After we air-dry and heat fix the endospore slide we add Malachite Green and then
Set on heated hot plate for 5 Minutes
How long do we let the malachite green cool after we take it off the 2nd hot plate?
3 minutes
What is the stain used in “negative stain”
Nigrosin
With the negative stain what were the 2 species we examined?
What was the alternative choice
B. Cereus & S. Epidermidis
Mouth biofilm
What steps do we skip in both the negative stain and the capsule stain?
Heat fixing
Rinsing with distilled water
Which is species is in the purple broth?
Which staining method is it used for?
Gram positive or negative
Morphology and arrangements
Enterobacter aerogenes / Klebsiella aerogenes
Capsule Stain
Gram negative
Diplobacillus
Which stain do we use Aqueous Crystal Violet for? 1 minute
Capsule stain
In the capsule stain we use which three reagents and for how long?
Nigrosin
Aqueous Crystal Violet 1 minutos
20% Copper Sulfate 3 minutos
What do we use to rinse the slide in the capsule stain?
20% Copper sulfate
Acid fast bacteria are also (gram-negative/ gram-positive)
Gram-positive
Name the 2 reagents used in endospore staining, in order
When do we add them
Do we rinse, if so with what?
after air dry and heat fixing, place on hot plate and add Malachite Green 5 minutes
Remove from hot plate and rinse with distilled water
Safranin Red & Rinse with distilled water
Kinyoun method aka
Reagents
Mordant
Bacteria we looked at, results Color
Acid-fast
Basic fuchsin
2ndary stain Methylene Blue
DSMO
Mycobacterium smegmatis = Acid fast Red
S. Epidermidis = non acid fast Blue
What are the colors and the species from the Schaeffer-Fulton method.
Name of species used
Red = vegetative cells
Green = endospores
Bacillus cereus
Exosporium does what what is it associated with?
Composition
Protective barrier for endo spore.
Proteins
What is the Mordant in the endospore staining
Heat
What is the 2ndary stain in the Schaeffer-Fulton endospore stain?
Safranin
Which species doesn’t produce endospores
Clostridium, Mycobacterium, Bacillus
Mycobacterium
Mycobacterium tuberculosis is visible under which kind of staining?
Results from this stain
Kinyoun Acid-fast
Pink
Advantage of Kinyoun staining over Ziehl-Neelsen?
Which did we use?
Kinyoun doesn’t involve heating releasing poison gas
Kinyoun
Name acid-fast genuses
Mycobacterium & Nocardia
What makes Mycobacterium resistant to staining
Mycolic acid
Describe E coli
Gram status
Morphology and arrangements
Negative (Pink)
Bacilli Single or Diplobacillus
Staphylococcus epidermidis before primary stain
Colorless
Pseudomonas fluorescens after primary stain?
Purple
Pseudomonas fluorescens
Gram status
Morphology and arrangements
Negative
Bacillus
Single
Bacillus megaterium after the addition of the Mordant
Purple
Bacillus megaterium
Gram status
Morphology and arrangements
Positive
Streptobacillus
Staphylococcus epidermidis after Decolorizer
Purple
Moraxella catarrhalis after Decolorizer
Colorless
Moraxella catarrhalis
Gram status
Morphology and arrangements
Negative
Diplococci
Pseudomonas fluorescens after Decolorizer
Clear
Bacillus megaterium after counter stain
Purple
Escherichia coli if you forget to add the counter stain
Clear
Escherichia coli if you forget to apply the Decolorizer
Purple
Bacillus megaterium if you forget to add Mordant (Grams Iodine)
Red
On the pipette pump where is 1ml?
At the 0 mark
0.1 ml is how much on the pipette pump
Between any 2 whole number lines
Standard plate count
1 ml from each test tube to the next filled with 9 ml
What is the dilution of A B C
A = 1: 100
B = 1: 10,000
C= 1: 1,000,000
1 ml from test tube B on the plate. What is the total dilution?
1:10,000
0.1 ml from testtube B on the plate has What total dilution?
1:100,000
1 ml from testtube C on the plate has what total dilution?
1:1,000,000
0.1 ml from test tube C has What total dilution?
1:10,000,000
What is the control in the standard plate count?
1 ml E. Coli
Simple staining is…
Using 1 dye to stain a bacterial smear
Type of attraction between cell and stain
Electrostatic
Describe shape of Fusiform bacteria
Which group of cell shapes do they fall into?
Bacillus
But with tapered ends
Staphylococcus aureus, Streptococcus lactis, Bacillus megaterium and Corynebacterium xerosis were examined by this staining type
Simple
Pleomorphism refers to
Demonstrating several different shapes
Distinct reddish purple granules with in a cell that show up with Methylene Blue
Metachromatic granules
3 things you can discern with a good smear
- Morphology (Rods, Cocci, Shapes)
- Arrangement ( Single, Stapho, Strepto)
- Internal structures ( endospores & cell inclusions)
Goals of a good smear
- Adhere cells so they are not washed off during the staining and rinsing
- Ensure shrinkage doesn’t happen to cells. ( causing Distortion & Artifacts)
- Prepare thin smears
For solid media when creating a smear which do you add first the smear or the water?
Water
Where do we write the name of the organism on a slide
Left-hand side
A hot loop will cause what to happen to the bacteria
They will go into the air
When do we flame the tube?
After opening
After putting the inoculation loop in
Protoslo does…
Slows movement of bacteria
Gram staining is which kind
Differential
