Exercise 6_ Bacterial Cell Wall Flashcards
primarily provides a protective shield to bacteria against hostile environment, allow communication with its surroundings to find optimal conditions for growth and development of the bacterial cell
bacterial cell wall
role of bacterial cell wall
- protective shield to bacteria against hostile environment, - allow communication with its surroundings to find optimal conditions for growth and development of the bacterial cell - its rigidity maintains the shape of the bacterium - enables transport of large molecules into and out of the cell
who developed gram stain
Danish scientist and physician, Hans Christian Joachim gram
when was gram stain developed?
year 1884
gram staining is what type of staining method
differential staining
advantages of using gram staining
- rapid turnaround and generation of information about the organism’s gram rxn - available inexpensive reagents
2 divisions of bacteria according to gram staining method
gram + gram -
what can be seen under the light microscope after staining the cells with gram stain
cell shape cell size cell arrangement
what differentiates the gram reaction of a bacterium when stained with gram stain
surface layer composition (cell wall)
Gram ____ are decolorized by alcohol, losing the purple color of crystal violet
negative
Gram ____ are not decolorized by alcohol, can retain the purple color of crystal violet
positive
True or false: a bacterium with a thick peptidoglycan can retain crystal violet after decolorization of alcohol
true
True or false: a bacterium with a thin peptidoglycan can retain crystal violet after decolorization of alcohol
false
counterstain medium of gram stain
safranin
what color does safranin impart to the cells of the decolorized gram negative bacterium?
red or pink
which gram bacterium will manifest red/pink colored cells after decolorization
gram negative
which gram bacterium will manifest violet or blue colored cells after decolorization
gram positive
what method can be used to determine the gram rxn of an unknown bacterium
gregersen’s method
if the bacterium mounted in KOH turns viscous it is gram
gram negative
if the bacterium mounted in KOH turns watery it is gram
gram positive
control organisms utilized in this exercise
B polymyxa E coli
gram negative control organism
E coli
E coli’s gram reaction
gram negative
gram positive control organism
B polymyxa
B polymyxa’s gram reaction
gram positive
percent concentration of KOH
0.03
mounting medium of gram stai
distilled water
mounting medium of gregersen’s method
3% KOH
3% KOH is the mounting medium of which method?
gregersen’s method
distilled water is the mounting medium of which method?
gram stain
During gregersen’s test, do you heat fix samples?
NO
During gram staining, do you heat fix samples?
yes
1st staining agent utilized during gram staining
crystal violet
time requirement in staining cells with crystal violet
1 min
after staining with crystal violet, what’s next?
wash smear with tap water
next stain after crystal violet
gram’s iodine
time requirement in staining cells with gram’s iodine
1-2 min
decolorizer of gram staining
95% ethanol
percent concentration of the decolorizer
0.95
time requirement in decolorizing cells
10-15 secs
counter stain used in gram staining
safranin
time requirement in staining cells with safranin
45 secs
where does crystal violet binds to?
peptidoglycan
gram’s iodine serves as the ____ which combines the stain with the peptidoglycan of the bacterial cell wall forming a complex
mordant (fixing agent)
false gram stain results can be because of
- excessive heat fixation - concentration of primary stain - smear is too thick - faulty smear prep - age of culture - use of antibiotics - insufficient iodine exposure - prolonged decolorization - excessive counterstain - lack of experience
this bacteria cannot be stained using gram staining due to the lack of a peptidoglycan in the cell wall
Mycoplasma
Mycoplasma cannot be stained by gram staining because of what
absence of peptidoglycan in the cell wall
the viscous sample in gregersen’s test contains
cell wall debris and DNA
the outer membrane of the gram-negative bacteria interact with ____ ____ causing the hydrolysis of the ____ bonds breaking down the cell wall including the peptidoglycan
hydroxide ions, ester
hydroxide ions interact with what part of the gram negative bacterium
outer membrane
what does the gram negative has that is not found in gram positive bacteria
outer membrane thick peptidoglycan
what aids the cell wall of gram positive bacteria to not be degraded during gregersen’s method?
thick peptidoglycan
as a control organism, what is expected to be the color of the cells of E coli after gram staining
pink/re
as a control organism, what is expected to be the color of the cells of B polymyx after gram staining
purple/blue
alternatives for crystal violet as the primary stain for gram staining
methyl violet/gentian violet
methyl violet/gentian violet can be alternative of what stain in gram staining method?
primary stain
staining alternatives for safranin as the counterstaining agent in gram staining
dilute carbol fuchsin or neutral red
dilute carbol fuchsin or neutral red can be alternative of what stain in gram staining method?
counterstain/safranin
effect of replacing iodine with other oxidizing agents (eg. bromine, chlorine/fluorine)
can cause decolorization unnecessary cell wall interaction not form complexes with the primary stain
importance of using freshly prepared KO
-KOH degrades over time as it reacts with the environment which can affect its ability to differentiate the gram positive and gram negative bacteria -Remains pure from contaminants (eg carbon dioxide)
KOH stands for
potassium hydroxide
higher concentrations than the 3% optimum concentrations of the KOH it will produce false ____ results
negative
lower concentrations than the 3% optimum concentrations of the KOH it will produce false ____ results
positive
