preparation + staining Flashcards
why do we stain bacteria
bacteria are colorless, transparent, and difficult to see
what is staining
coloring the microorganism with a dye for visualization
what can we see via staining
certain structures within the organism or the organism itself
what must we do to a microbe before staining it (1 word)
we must fix it
what is fixing
attaching the microbe to the slide, killing it, and preserving internal and external structures in their natural state
how is fixing done
a smear is made, the slide is air dried, and it is fixed by passing it through a flame several times
define smear
a thin film of bacteria on the surface of the slide
what are two ways we can make a smear
plate or broth
describe a plate smear
a drop of water is put onto the slide first, and then an inoculating loop is used to get the cells, then the cells are spread into the water
describe a broth smear
put inoculating loop into a broth, and then smear it onto the slide
describe the orientation of the slide when fixing it through a flame
smear side up
what two methods can be used for fixing microorganisms
heat fixation and chemical fixation
describe heat fixation
preparing the slide then passing it through a flame
describe chemical fixation
a fixative is added to the smear
in chemical fixation, give examples of fixative that can be added to the smear
ethanol, methanol, acetic acid, formaldehyde
which method of fixation (heat or chemical) is better for preserving the internal morphology of cells
chemical
describe the features of the dyes used to stain organisms
they’re ionic, and they have chromophore groups attached
what do chromophore groups do
give the dye its color
what two classes can ionizable dyes be divided into
basic and acidic dyes
what type of charged groups do basic dyes have
positive
what types of molecules do basic dyes bind do? give examples
negatively charged molecules
nucleic acids, many proteins
which type of dye (basic or acidic) is most used in microbiology? why?
basic; because they’re attracted to negative charges and bacterial cell surfaces are negatively charged
give examples of basic dyes
methylene blue, malachite green, crystal violet, safranin
what type of charged groups do acidic dyes have
negative
what types of molecules do acidic dyes bind to? give examples
positively charged things
some proteins
describe how acidic dyes interact with bacteria
they repel bacteria due to having the same charge
give examples of acidic dyes
eosin, acid fuchsin, congo red
what are the two types of staining procedures
simple staining and differential staining
what is simple staining
microbes are stained with a single agent (1 color)
describe the steps of simple staining
prepare smear, air dry, fix the bacteria, apply the dye, rinse off excess, blot dry, examine
what are the uses of simple staining
used to highlight the entire microbe, allows one to get info about the size, shape, and arrangement of cells
what is differential staining
used to divide bacteria into separate groups based on their staining properties
what are two types of differential staining
gram stain and acid fast stain
what is a gram stain
divides bacteria into two groups based on their ability to take up and retain a dye
what two groups are made in a gram stain
gram positive and gram negative
what type of smear is used in a gram stain; heat or chemical
heat fixed
describe the steps of a gram stain
apply crystal violet dye, wash, then apply iodine = everything purple
rinse, use ethanol for decolorization (gram negative will be colorless)
wash, apply safranin (counterstain), turns colorless into pink
what dyes are used in a gram stain
crystal violet and safranin
what are the results of the gram stain
gram negative = red
gram positive = blue
what is an acid stain used for
used to stain a small group of organisms that do not readily take up stains + differentiates between 2 groups of bacteria
what two groups of bacteria are defined in an acid fast stain
acid fast vs non acid fast
define acid fast organism
organisms that do not readily take up stains due to high content of lipid in their cell walls
what type of smear is used in an acid fast stain: chemical or heat fixed
heat fixed
describe the steps of an acid fast stain
add primary stain (carbofuchsin) while slide is over steaming water
cool + rinse, add decolorizer then acid alcohol
wash, then apply counterstain (methylene blue)
in an acid fast stain, why do we apply the primary stain while the slide is over steaming water
the heat is used to push the dye through the cell wall since acid fast organisms dont readily take up dye
what two dyes are used in an acid fast stain
carbofuchsin red and methylene blue
what are the results of the acid fast stain
acid fast = red
non acid fast = blue
define endospore
a resistant structure that allows some bacteria to withstand unfavourable conditions
what is the name for the procedure to stain an endospore
schaeffer-fulton procedure
describe how we stain an endospore
using a heat fixed smear, add malachite green in the presence of steam (this will penetrate the endospore)
cool + rinse, add safranin as the counterstain, wash
what are the visual results of the endospore stain
endospore=green
cytoplasm=red
what does the endospore stain allow us to see
presence of an endospore + location of the endospore
what are flagella used for
locomotion of bacterial cells
what do we do for a flagella stain
add carbolfuchsin
why would we want to do a flagella stain
aids in identifying bacteria based on presence and location of the flagella
what is a negative stain
its when we stain the background instead of the organism
what color will bacteria be after a negative stain
colorless; only the background is stained
what dye is used in a negative stain
an acidic dye, ie eosin
what is a negative stain used for
revealing the presence of a capsule
what is a capsule
a substance that may surround some bacterial cells
what is the charge on a capsule
negative
