Post Laboratory Bacterial Morphology Flashcards
rigid structure which gives shape to the cell; prevents cell from bursting when present in a hypotonic environment
cell wall
rigid portion of cellwall is known as (3)
murein
peptidoglycan
mucopeptide
varies in chemical composition and structure from species to species
peptidoglycan
cell wall is composed of
NAG
NAM
amino acid side chains
NAG
N-acetylglucosamine
NAM
n-acetylmuramic acid
with regards to PG content, bacteria can be divided into 2 groups:
gram +
gram -
comparison of PG of G+ and G-, identify which is which
50%
10%
G+ - 50%
G- 10%
comparison of lipids of G+ and G-, identify which is which
0.2%
10-20%
G+ 0.2%
G- 10-20%
comparison of polysaccharides of G+ and G-, identify which is which
35-60%
15-20%
35-60% - G+
15-20 G-
acidic dye used in CW staining, negatively charged
congo red
basic dye used in CW staining; positively charged
methylene blue
cationic mordant which causes reversal of surface charges
cetylpyridinium chloride
s used as a staining agent for bacterial cell walls due to its cationic nature, which allows it to bind to the negatively charged components of the cell wall, effectively highlighting its structure under a microscop
cetylpyridinum chloride
result when congo red, methylene blue, and cetylpyridinum is used for cw staining
blue cytoplasm
red cell wall
use of four different reagents to differentiate the two types of bacteria based on cell wall composition
gram staining
primary stain for gram staining
ammonium oxalate crystal violet
basic dye for gram staining to color cell constituent
ammonium oxalate crystal violet
mordant used for gram staining
gram iodine
use of mordant
stain affinity enhancer
form relatively insoluble complex with primary stain
decolorizing agent for gram staining
95% ethanol
removes dye from stained cells
decolorizing agent
basic dye used to give color to decolorized cell
safranin
used for confirmation of gram reaction using KOH
Gregersen’s method
make use of strong base/alkali to cause lysis of G- CW
gregersen’s method
: layer of gelatinous material or viscous substance produced or secreted by many
bacterial cells which accumulates around the cells and coat the cell wall
capsule
Most capsules are
composed of polysaccharides called as
exopolysaccarides
capsules composed of polypeptides are called
exopolypaptides
e usually demonstrates a
specific shape
capsule
r has a stringy unstructured appearanc
slime layer
chemical composition of capsule
polysacchride
protein (peptide)
protein-polysaccharide complex
es: non-ionic: HzO-soluble; glucose, amino sugar component of capsule
polysaccharides
example of polypeptide in capsule
glutamic acid
protein-polysaccharide complex present in protein
glycoprotein
are not essential for survival but advantageous
capsules
importance of capsule where H2O content of cell increases
protection from dessication
importance of capsule where it resist phagocytes
Ability to resist phagocytos
capsules also blocks the attachment of ___
bacteriophages
forms plaque on teeth; attachment strengthened by
presence of glucans/glucose sub-units; synthesized only from sucrose a.s substrate
streptococcus mutans
The size or thickness of capsules is influenced by the
environment or medium where
organisms grow
Best method to observe capsules is the use of
phase contrast microscopy
view Cell components with different refractive index, Each structure will undergo a change in phase, translated into brightness of
structures, detectable by the human eye
phase contrast microscopy
Abihty to produce capsules is a ___traiU but production is affected by the kind of
medium in which cells are cultivated (___ ___)
trait
environmental factor
. In some strains, capsules formation is indicative of ___
virulence
. All bacterial capsules are ___soluble
water
chemical composition of capsule formation can vary between
polymers of glucose
polymers of amino acid
polypeptide
polysaccharide protein complex
glucose polymers for capsule
dextran
glucose polymers for amino acid
hyaluronic acid
glucose polymers for polypeptides
d-glutmaic acid
negative staining techniques for capsules staining (2)
duguid’s method
maneval’s method
structural staining for capsule staining
structural staining
: indirect simple staining
o Technique by which without staining, cells are made readily visible in an
otherwise dark background
negative staining
negative staining d: made use of nigrosin
duguid’s method
Coloring portion of dye bears a negative (-) charge; does not stain cells
due to repulsion of charges
. Fails to stain cells, instead, forms deposits around the cells
. Advantages: : r gives an accurate view of the cell
r cells do not receive vigorous chemicaVphysical treatment
. very simple
what staining for capsule
duguid’s method
stain of maneval as a pH indicator
congo red
maneval’s solution is made up of
phenol
acetic acid
ferric chloride
acid fuschin is made up of ___ and __
phenol
ferric chloride
cells are stained, capsules colorless, and backgrond is blue/red
what staining for capsule
maneval’s staining
causes lowernig in pH in maneval
acetic acid
previously applied –+ detect
change in pH, red to blue, observe cells, capsules better
congo red
o Special staining method available for demonstration of structures within or exterior to the cell wall
anthony’s method
based on the fact that structure exhibits different degree of affinity
for dye from rest of the cell, making differentiation possible
anthony’s method
: basic dye, stains the cell, but not capsule for anthony’s method
crystal violet
used to emulsify cells in maneval’s staining
glucose solution
replaces HzO for washing; so that capsule will not be
dissolved (capsules: non-ionic)
copper sulfate
Reaction with ___and saccharides: ___ reacts with
saccharides, retained in capsule (with color)
copper
less capsule formed due to lower sugars concentration
what media
NAG slant
greater capsule formation due to higher sucrose concentration
what media
NAS
Polysaccharide capsule produced best on ___ media and not from
hexoses
sucrose
: more effective precursor: glycosidic linkage already
formed, can be transferred to other sugar units with little or no
expenditure of energy
sucrose
Many microorganisms store reserve food materials as ___in the cell when cultures are
grown on media with limiting nutrient
granules
Reserve materials can be classified into 3 large chemicalgroups:
intracellular lipids
polysaccharides
polyphosphate granules
composed of polymerized poly-B-hydroxybutyric acid (PHBs) or sudanophilic granules
intracellular lipids
intracellular lipids are composed of
polymerized polyhydroxybutyratic acid (PHB) or sudanophilic granules
formation of __is common in bacteria
PHBs
a; formed when C and energy sources are in
excess
PHB
- accounts for as much as 50”/” cell’s dry weight
intracellular lipids
intracellular lipids have high affinity to fat soluble dyes such as
sudan black B
k has strong affinity to lipids such that xylene treatment is unabb to extract the
dye;
sudan black
Sudan black has strong affinity to lipids such that ___treatment is unabb to extract the
dye
xylene
lesser affinity between the Sudan black and other cell components causes the dye to
be removed during ___treatmen
xylene
produced in response to excess C when N, S or P are limiting or when pH is low
polysaccharide (glucose polymers)
- appear at early log phase and become more numerous as the culture ages
polysaccharide (glucose polymers)
- accounts for as much as 15% of the cell’s dry weight
polysaccharide (glucose polymers)
: contains 20% water soluble fraction amylose (linear polymer) and 80% water
insoluble fraction amylopectin (branched polymer
starch
starch contains ___ as a 20% water soluble fraction (linear)
amylose
starch contains __ 80% water insoluble fraction (branched)
amylopectin
lodine treatment causes complexing of iodine with amylose and amylopectin
forming a structure which exhibits strong absorption of __ - dark blue to
blue-black
light
color of iodine with amylose and amylopectin
dark blue to blue-black
- l2 is being trapped in the ___ structure of amylose and amylopectin
a-helical
contiains extensive branches (amylopectin)
glycogen
lz can also be trapped but because of extensive branching (o-1,6linkages)
and less linear helical structure, reaction is weaker resulting to pale color
reactio
glycogen
glyocgen reacting with iodine
reddish brown
metachromatic granules or volutin
polyphosphate granules
- rnay constitute 40-50% of total cell phosphorus
polyphosphate granules
composed primarily of polyphosphorus with trace amounts of RNA, DNA and proteins
polyphosphate granules
- formed at the log phase of growth when P is in excess
polyphosphate granules
polyphosphate granules are - formed at the ___ phase of growth when P is in excess
log
- functions as a phosphagen: energy storage and phosphate reserve
POLYPHOSPHATE GRANULES
possess numerous negative
charges with stronger affinig to basic dyes than other cell component
polyphosphate
Other granules composed of inorganic compounds which €n be formed by the
microorganisms
sulfur globules
- present in bacteria which oxidize sulphide
sulfur globules
organisms oxidize H2S shancing it to ___globules deposited either inside or
outside the cell
elemental sulfur
g, make use of this globule and oxidize it further to
sulfates
sulfur globules
Condition for Formation : Low N but with available C and Energy sources
storage granules
due to low concentration of this, protein and nucleic acid synthesis is low so cell accumulates excess amounts of C and energy sources
nitrogen
when nitrogen increases, ___ are used for metabolism
reserve granules
single 20 to 80 nm thick layer of peptidoglycan (murein) lying outside plasma membrane
what bacteria in CW staining
gram positive
has a 2 to 7 nm peptidoglycan layer covered by a 7 to 8 nm thick outer membrane (quite complex)
gram negative bacteria
all the structures from the plasma membrane outward (plasma membrane, cell wall, capsule)
cell envelope
space between the plasma membrane and the outer membrane of gram negative bacteria, and is sometimes also observed between the plasma membrane and the wall in gram positive bacteria
periplasmic space
substance that occupies the periplasmic space
periplasm
usually large amounts present in G+ CW, covalently connected to PG/PM
teichoic acid
extends to the peptidoglycan surface
being negatively charged, give the cell wall its negatively charge
teichoic acid
teichoic acids are polymers of __ joined by phosphate groups
glycerol
ribitol
lies between plasma membrane and cell wall; contain few proteins usually attached to PM
periplasm
involved in interactions of cell with its environment, bind to PG, TA or other receptors either covalently or noncovalently
surface proteins
example of surface proteins in G+
s-lyaer
m-protein
salient features of g+ cw (4)
peptidoglycan
teichoic acid
periplasm
surface proteins
salient features of g- cw (4)
peptidoglycan
outer membrane
porin proteins
periplasm
lies outside thin PG layer, linked to cell by Braun’s lipoprotein and many adhesion sites
outermembrane of G- cell
component of outer membrane that has core polysaccharide and o-side chain for endotoxin, adhesion, virulence, and permeability barrier
lipid
make OM more permeable in G- than plasma membrane by permitting passage of smaller molecules like glucose and other monosaccharides
porin protens
lies between outer and inner membrane in G-, contain periplasmic proteins that help in nutrient (e.g. hydrolytic enzymes) and transport proteins
periplasm
- 3 layers
- no periplasmic space
- peptidoglycan is 5 to 10 nm thick
- unique outer cell membrane
- teichoic acid absent
- murein lipoprotein
what CW
gram negative
- peptidoglycan 20 to 80 nm
- lipopolysaccharide
- teichoic acid (antigenic)
- other ptoeins
what wall
gram positive
In aqueous solutions, crystal violet dissociates into __ and ___ ions, penetrates through the cell wall and membrane, resulting to purple color.
CV+
CI -
as mordant interacts with CV+ to form large CV-I complexes within the cytoplasm and outer layers of cell.
iodine
either ethanol or acetone, interacts with lipids of membranes. The outer membrane loses from the cell, resulting to exposed peptidoglycan.
decolorizer
In Gram ___bacteria, ethanol dehydrates peptidoglycan, traps the large CV-I complexes, so it remains purple in color
positive
In Gram ___bacteria, ethanol makes the membrane to be leaky, allowing the large CV-I complexes to be washed from the cell, thus losing purple color.
negative
is the most crucial step, over-decolorizing may lead to erroneous result (Standard: 15 sec)
decolorizing
is the most rapid decolorizer
acetone
is recommended to prevent over decolorization of samples
ethanol
when overexpose to cells of Gram positive bacteria, may replace CV dye, although mordant slows the process(Standard: 30 sec-1 min)
counterstain
may influence stain results, as older cultures may have breaks in the cell wall and thus resulting into gram variable results
age of culture
may be used as confirmatory test, here formation of string (DNA) indicates the isolate is a Gram negative bacteria. Gram positive bacteria does not produce a string (unaffected
(potassium hydroxide test)
list of gram positive bacteria
Staphylococcus aureus
Streptococcus pneumoniae
Enterococcus faecalis
Bacillus anthracis
Corynebacterium diphtheriae
Clostridium botulinum
Mycobacterium tuberculosis
Bacillus megaterium
Bacillus subtilis
Bacillus cereus
list of gram negative bacteria
Escherichia coli
Vibrio cholerae
Salmonella enterica
Pseudomonas aeruginosa
Klebsiella pneumoniae
Neisseria gonorrhoeae
Resulting to a mix of pink and purple color, due to:
gram variable
species where Cell wall is sensitive to breakage during cell division
actinomyces, anthrobacter, corynebacterium, mycobacterium, propionibacterium
Decrease in peptidoglycan thickness during growth gram variability in
bacillus
butyrivibrio
clostridium
resistant to decolorization step, appear as Gram positive
acinetobacter (gram negative)
)- waxy nature of the coat results to non-Gram stainable
mycobacterium (Gram positive)
instead, mycobacterium is stained using
an acid-fast stain, most commonly the Ziehl-Neelsen stain
– unusual cell wall structure appear to stain Gram negative or Gram variable
gardnella (gram positive)
– ammonium oxalate is added to prevent dye precipitation and uses alcoholic solution of counterstain
what modification in gram staining
hucker’s method
sodium bicarbonate is added to crystal violet; prevents the acidification of the solution as iodine oxidizes; uses the aqueous solution of safranin
what modification in gram staining
burke’s method
where is capsule synthesized and secreted to the outside of the cell where it surrounds the bacterium
capsule
most of the capsulated bacteria have a capsule made up of ____ but some have __ or __
polysaccharide
polypeptide
glycoprotein
is a type of differential stain which uses acidic and basic dyes to stain background and bacterial cells respectively
capsule stain
method where india ink and crystal violet dyes are used
india ink method
to demonstrate ___, india ink method
cryptococcus
in this type of capsule stianing procedure, the primary stain is crystal violet and all parts of the cell take up the violet stain
there is no mordant but a 20% copper sulfate solution serves as decolorizing agent and counterstain
what method
anthony’s stain
there is no mordant but a 20% ___ ___ serves as decolorizing agent and counterstain
copper sulfate
it decolorizes the capsule by washing out the crystal violet but will not decolorize the cell
copper sulfate solution
if stained with copper sulfate, the capsule appears as a faint __ halo around a purple cell
blue
either gram positive or gram negative – surrounded by
glycocalyx
polysaccharide containing layer)
of capsule
glycocalyx
when the layer’s composition is tightly bound and remained attached to cell
capsule
when the layer is loosely-bound, difficult to see, and do not exclude particles
slime layer
smooth appearance is agar media (described as “smooth”)
encapsulated bacteria
rough appearance is agar media (described as “rough”)
non encapsulated bacteria
Capsule – composed of polysaccharides; may also contain
polyalcohol and polyamines
Role in pathogenicity, acting as virulence factors to protect cells from phagocytosis and/or complement-mediated ex
pseudomonas
rhizobium
highly hydrated (95% water), so not to heat-fixing (will cause distortion and shrinking of the capsule)
capsular polysaccharides
– interacts with the negative ions of the bacterial cell
basic dye
causes the precipitation of the capsular material (ex. Metal ions, alcohol, acetic acid)
mordant
will remove unheat fixed bacteria and may cause capsule disruption
blotting
In some preparations, cells are exposed to antibody against capsular ___to enlarge the capsule
antigens
no visible capsule because no milk or other proteinaceous material was present or added to CV and provide a background
kliebsella pneumoniae
When rinsing, it should be done with care, shaking the media (shaking of tube) will shake ___ ___
slime layers
Capsules are seen better at the ___of the slide
edges
– used as primary stain (interacting with protein material in culture)
crystal violet
serves as mordant and decolorizer agent in anthony’s staining
copper sulfate
maenval’s solution is a mixture of
acetic acid
acid fuschin
main stain that interacts with the bacterial cell, staining the cell bright red
fuschin
– lowers pH, serves as mordant (stabilizes capsule)
aceitc acid
– counterstain, pH indicator (blue for pH below 3.0, red for pH above 5.2), dries to form a red background
congo red
color of congo red when pH is below 3.0
blue
color of congo red when pH is above 5.2
red
dries to form a red background
congo red
LIST OF ENCAPSULATED BACTERIA
Yersinia pestis
Streptococcus pneumoniae
Klebsiella pneumoniae
Bacillus anthracis
Haemophilus influenza
Pseudomonas aeruginosa
Neisseria meningitis
Cryptococcus Neoformans
Acetobacter
Bacillus cereus
has high molecular weight and osmatically inert
storage granules
storage granules include (3, ppt)
polymetaphosphate
poly-b-hydroxybutyrate
polyglucan
stored in form of line or chains of inorganic pyrophoshpate
phosphate
when nucleic acid synthesis is prevented by starvation, granules accumulate in the ___
cytoplasm
granules stained with sudan black
poly-b-hydroxybutyrate, lipid granules
PHB granules are ___
polyactides
formed by condensation of acetyl coA
PHB
it may make up 60% of dry weight in bacteria like ___ after growth on acetate and butyrate
b. megaterium
these polyglucan granules are found in what genera
clostridia and coliforms
resembles glycogen of mammalian liver in structure
polyglucan
polyglucan granules are also found in
protozoa
yeast
fungi
algae
staining method for storage granules where the granles are bluish black and organism is green
albert’s staining
staining method where granules are deep blue and organism is pink
neisser’s stain
s a technique used to identify and observe metachromatic granules in Corynebacterium diphtheriae. This bacterium causes diphtheria. Albert’s staining is a differential stain that uses two reagents, Albert’s A and Albert’s B.
albert’s staining
stains the bacillus green
what dye of albert’s stain
albert A
stains the granules bluish black
what dye of albert’s stain
albert B
is a microbiological stain for visualising the polar bodies in the cytoplasm of gram-positive bacteria. This staining is used to identify diphtheria bacteria.
neisser’s stain
present in cytoplasm of centrain bacteria and first time observed in spirillum voluntans
volutin granules
means abiity to change color
volutin granules
bacteria with volutin granules
lactobacilli
corynebacteria
desulfovibrio
requirement for albert’s staining
clean grease free slide
nichrome wireloop
24 hours of culture
albert’s stain
albert’s iodine
xylene (if curd sample is used)
albert’s stain is made up of
malachite green
toludine blue O
acetic acid
check albert stain procedure (slide 50)
+1
separates bacteria in two groups (neisser positive and negative)
neisser stain
distinguishes those filaments with ells that contain granules hat accumulate phosphate
neisser stain
neisser positive is colored
blue
neisser positive is colored
brown
result of cytoplasm and granules in neisser’s stain
pink - cytoplasm
deep blue - granules
check neisser’s stain slide 55
+1
takes place when certain negatively charged groups on the cell react with cationic dyes; on polymerization the original color of dye changes to another color
metachromasia
constituent of metachromatic granules
polymetaphosphate
reserved source of food, associated with cytoplasmic granules, formed during nutrient imbalance
polymetaphosphate
Chiefly used to demonstrate metachromatic granules (Ex. Corynebacterium diphtheriae)
albert’s staining
– consists of toluidine blue, malachite green, glacial acetic acid, and ethyl alcohol
albert A
are basic dyes with high affinity for acidic components for albert’s staining
toluidine blue
malachite green
The pH of Albert stain is adjusted to ___ by using acetic acid; becomes basic for volutin granules as their pH is highly acidic
2.8
The pH of Albert stain is adjusted to 2.8 by using acetic acid; becomes basic for___granules as their pH is highly acidic
volutin
On applying Albert’s stain to the smear, ___) stains volutin granules and ___ ___ stains the cytoplasm (blue-green)
toluidine blue
malachite green
contains iodine and potassium iodide in water
albert B solution
On adding this solution, due to the effect of iodine, the metachromatic property is not observed and granules appear blue in color.
albert B solution
due to albert B solution, bacterial cell stain __ and granules stain __
green
black
In observation of ___, it appeared as green-colored, rod-shaped bacteria that are arranged in angles to each other resembling L and V or Chinese letter pattern with bluish black metachromatic granules at the poles.
bacillus
Staining according to Neisser is a test for the presence of ___stored in the cells (storage materials).
polyphosphate
This method is an indispensable aid to the identification of certain strains of filamentous bacteria.
neisser’s staining
Furthermore, this staining method can make the Bio-P bacteria, responsible for biological phosphate removal, visible.
neisser’s staining
necessary solutions for neisser’s staining (slide 61)
+1
neisser stianing procedure
·Prepare a fixed smear.
·Place a freshly made mixture of 2 parts solution A and 1 part solution B onto the slide for a contact period of 10-15 seconds. Afterwards, allow the excess dye to run off the slide.
·Add solution C for a contact period of 45 seconds.
·Rinse the slide with tap water (with the flow against the back of the slide).
·Allow the slide to dry and then view with a 100x bright field objective. Drying can be speeded up by removing most of the water carefully with filter paper.
stain hardly or not at all (slightly brown or yellow)
what result of neisser’s staining
negative
In filamentous bacteria, contain blue-black colored polyphosphate globules
what result of neisser staining
positive
In ____bacteria, contain blue-black colored polyphosphate globules
filamentious
Colonies of blue-black coloured cells. These are comprised of ___ bacteria.
bio-P
