lab practical Flashcards
parts of a microscope
ocular lens nosepiece objectives stage slide holder condenser diaphragm light source lock screw coarse and fine adjustment mechanical stage control
what function does the condenser have?
focuses light onto the specimen
what function does the diaphragm have?
changes the intensity of the light
equation for resolution
wavelength of light / (2 x numerical aperture)
define numerical aperture
how far away they need to be from each other to be able to see 2 separate things (want the smallest number)
why do we use oil immersion?
in order to capture all the light and not let it escape
how do you get maximum resolution for a lens system?
correct alignment & adjusted to focus & oil
cleaning tissue used for microscope
lens paper
solvent used on a microscope
alcohol
how can you extend lamp life?
keeping oil out
brightfield microscopy
live & preserved stained specimens
specimen is dark, field is white
provides for cellular detail
dark field microscopy
live unstained specimens
specimen bright, field black
provides outline of specimen with reduced internal cellular detail
phase contrast microscopy
live specimens
contrasted against gray background
internal cellular detail
fluorescence microscopy
illuminates specimen
antibodies with fluorescent dyes emit visible light only if antibody recognizes and binds to cell
diagnostic tool
transmission electron microscopy
best resolution
to view internal structure of cells and viruses
for small specimens
scanning electron microscopy
produce 3d image
surface of cell
for small specimens
smear prep
heatfix, in order to ensure microorganisms are attached to slide and will not wash off during staining. cells are also killed in the process.
problem- may distort cells
simple staining (positive)
chromophore is positively charged, on inside of cell
requires heat fixing
need oil
distorted
negative staining
chromophore is negatively charged, on outside of cell
requires NO heat fixing
no oil
better shape
how does a smear prep of cells from a liquid medium differ from prep of cells from a solid medium?
must add a drop of water to slide before adding solid medium
what causes a stain to adhere to bacterial cells?
charge attraction
what is the difference between basic and acidic dyes?
Basic dyes have a positive charge and acidic dyes have a negative charge.
what are chromophores?
color bearing ions
shape of: bacillus
rod
shape of: coccus
spherical
shape of: spirochetes
spring
shape of: spirilla
cork screw
shape of: coccobacillus
short rods
shape of: vibrios
curved rod
arrangement of: strepto
chains
arrangement of: staphylo
clusters
arrangement of: tetrads
packets of 4
arrangement of: sarcinae
packets of 8, 16, 32
arrangement of: palisade
ununiformed clusters
advantages and disadvantages to a negative stain
not kiling cells
cant differentiate between cells
advantages and disadvantages to a positive stain
can use oil immersion
kill cell
Gram stain colors: postive crystal violet gram's iodine ethyl alcohol safranin
crystal violet: purple
gram’s iodine: purple
ethyl alcohol: purple
safranin: purple
Gram stain colors: negative crystal violet gram's iodine ethyl alcohol safranin
crystal violet: purple
gram’s iodine: purple
ethyl alcohol: white
safranin: pink
gram positive
coccus
gram negative
rod, more disease because thinner peptidoglycan layer
why is the gram strain considered a differential stain?
differentiates between two kinds of bacterial cells based on their cell wall structure and composition
how do gram positive and gram negative bacteria differ in cellular structure? how does this contribute to their differential staining properties?
a gram positive cell- has a thick cell wall, retains the crystal violet dye better in the presence of a decolorizer gram negative cell- which has a thin wall
How does the age of a culture affect the Gram stain reaction? What is an optimum culture age for a valid Gram reaction?
old cultures of gram positive cells may not retain stain as well as younger cultures and could give false negative results. 16-18 hours are best
Which step in the Gram stain procedure is most prone to error? If done incorrectly, how might that step affect the end result?
The decolorizer step is very important because it is the step in which the cells become differentiated (gram-positive cells are purple and gram-negative cells are colorless). If too much decolorizer is used, gram-positive cells will lose the primary stain and be counterstained pink. If too little decolorizer is used, gram-negative cells will not lose the primary stain and will remain purple after counterstaining.
In what type of cell, gram positive or gram negative, would you find lipopolysaccharides in its cell wall?
gram negative
what is the function of a mordant? and which reagant serves this purpose in the gram stain procedure?
it causes the primary stain to adhere better or be taken up by the cell so that it is not removed during the decolorizing step. Gram’s Iodine
if you omit the mordant step while gram-staining Staphylococcus areus, what color will it stain?
pink
if you omit the decolorizing step while gram-staining Escherichia coli, what color will it stain?
purple
What are the functions of endospores in bacteria?
Endospores are resting stages that allow bacteria to survive conditions unfavorable for growth.
what is the mordant in the spore stain?
boiling, It causes the endospore to expand allowing stain to penetrate the structure
what unique chemical compound is important in the heat resisitance of endospores?
calcium dipicolinate is found only in endospores and not in vegetative cells. it is associated with heat resisitance
what conditions are necessary to destroy endospores? in what device are these conditions achieved?
endospores must be exposed to 121 degrees C, 15 pounds of pressure for at least 15-20 mins. these conditions are achieved in the autoclave
identify some spore-forming bacteria that may be responsible for disease
Clostridium, Bacillus
clostridium tetani, Bacillus anthracis
Escherichia coli
gram reaction: gram negative
shape: bacillus
arrangement- none
Staphylococcus epidermidis
gram reaction: gram postive
shape: cocci
arrangment- staphylo
advantages and disadvantages to standard plate count
can see living cells
time consuming
equation for calculating the number of cells in culture tube
number of bacteria per ml = number of colonies X dilution factor
equation for total number of cells in original culture tube
total number of cells in original culture = number of bacteria / ml X volume of broth in culture tube
advantages and disadvantages to turbidity
quick
see both living and dead cells
methylene blue
simple stains/ postive
malachite green
spore
crystal violet
gram stain
india ink
negative staining
if forget mordant
pink
Staphylococcus aureus (gram type, shape)
gram-positive, coccus
sugar fermentation reactions
- can the organism break down certain sugars
- does it produce acid and/or gas
- phenol red
- fermentation tubes catch gas
- positive: yellow (orange- slow)
- negative: red or pink
mixed acid fermentation
- determines if mixed acids are produced in the presence of glucose
- methyl red
- MR-VP test tube
- positive: red
- negative: remain yellow
Voges Proskauer test
- determines if butanediol (acetylmethylcarbinol or acetoin) is produced by the fermentation of glucose
- MR-VP tube &broth
- reagent A&B
- positive: red within 5 mins
- negative:
Simmons Citrate test
- determines if bacteria can use citrate as sole source of energy and carbon
- citrate agar slant (bromothymol blue)
- positive: blue
- negative: green
catalase test
- indicates the presence of catalase in an organism (enzyme)
- dribble H2O2 down slant of agar to see bubbling (reagent)
- positive: bubble (gas produced)
- negative: no bubbling
nitrate reduction
- determines if bacteria can reduce nitrate to nitrite or other nitrogenous compounds
- A & B reagent
- reduced: pink/red
- add reagent C (Zn dust) if no color develops
- negative: pink/red, clear: positive
urea hydrolysis
- detect the presence of enzyme urease (degrades urea to ammonia)
- phenol red (reagent)
- positive: pink
- negative: orange
tryptophan degradation
- determines presence of enzyme tryphtophanase (converts tryptophan into indole and pyruvic acid)
- reagent: Kovac’s reagent
- positive: red layer at top
gelatin
- detects presence of gelatinase (degrade gelatin from solid to liquid)
- positive: liquid after ice bath
- negative: solid
O2 requirements
- thyglycolate test
- obligate aerobes: growth at top
- obligate anaerobes: growth at bottom
- facultative anaerobes: heavy growth at top, throughout
- aerotolerant anaerobes: equal growth
- microaerophiles: growth in middle
motility
- determines if bacteria is motile
- wet mount: look for movement under microscope
- stab method: use needle and stab agar
- motile: swim away from stab line
- non-motile: only grow along stab line
oxidase test
- indicates the presence of cytochrome oxidase (enzyme)
- Tryptic soy agar plate
- Becton Dickinson (oxidase reagent dropper)
- positive: purple under 30 seconds
mannitol salt agar
- selective and differential
- if significant growth- Staphylococcus because it can handle high salt (gram-positive)
- plate from red to yellow
levine EMB agar
- selective and differential
- purple: bacteria can ferment sucrose and lactose
- non-fermenters: normal colored colonies
- positive: dark red/brown-green
- gram-negative
