Bacterial Morphology Part 2 Flashcards
Certain species of bacteria such as Bacillus and Clostridium produce a dormant cell called
endospore
Certain species of bacteria such as __and __produce a dormant cell called endospore
Bacillus and Clostridium
Because of a complex and effective structural organization of the spore, it is highly resistant to adverse conditions like
high temperature
toxic chemicals
radiation
dessication
The capability for persistent survival is due to an outer membrane which is surrounded by the densely packed ___coat and ____, containing amyloid or amyloid-like proteins.
endospore coat
exosporium
The capability for persistent survival is due to an ___ ___ which is surrounded by the densely packed endospore coat and exosporium, containing amyloid or amyloid-like proteins.
outer membrane
The capability for persistent survival is due to an outer membrane which is surrounded by the densely packed endospore coat and exosporium, containing ___or ___-like proteins.
amyloid
is a waxy, insoluble protein that forms deposits in organs and tissues
amyloid
However, their presence can be demonstrated by certain staining procedures like the use of ___ ___ with gentle steaming
malachite green
sually ___is necessary to have well stained spore preparation
steaming
A rapid method for the detection of the presence of endospores is to stain the ___contents of the endospore coat and exosporium, the very surface components that make the coats of these endospores impenetrable
amyloid
the very surface components that make the coats of these endospores impenetrable
amyloid
When a bacillus endospore suspension is incubated with an amyloid staining dye _____ (ThT) under ambient conditions, ThT accumulates on the surface region of the endospore
thioflavin T
This allows the enhancement of the fluorescent images; thus, a good ___microscope is necessary when viewing ThT incubated bacillus endospore suspension
fluorescent
The use of ___ provides an effective and rapid means of staining endospores without any pre- or post-treatment of samples but has to be coupled with fluorescence microscopy.
thioflavin T
When conditions are favorable, endospores may undergo ___- a process that transforms an endospore to a vegetative cell.
germination
factors that favor endospore germination (4)
optimum moisture
appropriate nutrients
appropriate pH
appropriate temperature levels
The ability of an organism to move by itself is called
motility
Motility is closely linked with ____, the ability to orientate along certain chemical gradients.
chemotaxis
___ cells can move by means of different locomotory organelles such as cilia, flagella, or pseudopods.
eukaryotic
Eukaryotic cells can move by means of different locomotory organelles such
cilia
flagella
pseudopods
___ move by means of propeller-like flagella unique to bacteria or by special fibrils that produce a gliding form of motility
prokaryotes
Prokaryotes move by means of propeller-like ___ unique to bacteria or by special ___that produce a gliding form of motility
flagella
fibrils
Prokaryotes move by means of propeller-like ___unique to bacteria
flagella
produce a gliding form of motility in prokaryotes
special fibrils
also function as sensory organs as they are sensitive to various chemicals and environmental conditions
flagella
Flagella are also involved in attachment and can furthermore act as ___elements, enabling bacteria to overcome unfavorable surface topographies
structural
flagella synthesis is affected by growth conditions and physical factors such as
temperature
pH
presence of ions and oxygen
Almost all ___bacteria and about half of the ___are motile,
spiral
bacilli
shape of bacteria that has no motility
cocci
are actually below the visual limit in size; electron microscopy has shown that they are around 20 nanometer thick hollow tubes
flagella
Flagella are actually below the visual limit in size; electron microscopy has shown that they are around ___ nanometer thick hollow tubes
20
. Until such time that electron microscopy will be a standard technique for flagellar examination, flagella ___is practiced
staining
The principle applied is the introduction of ___of the preparations before staining.
mordanting
The use of special stains increases their apparent ___and makes them visible under the light microscope.
diameter
staining procedures for flagella (3)
Gray’s flagella stain,
Liefson
Modified Baileys
___methods are employed for motility determination depending on the pathogenicity of the organisms.
three
For ___, there are two slide techniques that one might use
nonpathogens
For ___, tube method can be used to stain motility
pathogens
For pathogens, ___method can be used to stain for motility
tube
are the common types of preparations for observing bacterial motility.
hanging drop technique
wet mount technique
the depression slide and cover slip allow the suspension of the microorganisms in a clear drop of water, saline solution, or broth.
what method for motility
hanging drop
, the plain slide and cover slip serve to protect and render the specimen easily visible what method for motility
wet mount
The specimen is enclosed between plane parallel surfaces so that it is substantially in one plane and is completely surrounded by the mounting medium.
what method of motility
wet mount
When working with pathogenic microorganisms such as the ______ , it is too dangerous to attempt to determine motility with slide techniques.
typhoid
. The procedure is to inoculate a tube of ___ or ___medium that can demonstrate the presence of motility for pathogenic bacteria
semisolid
SIM medium
SIM medium is also known as
Sulfur, Indole, Motility Media
This is a differential medium. It tests the ability of an organism to do several things: reduce sulfur, produce indole and swim through the agar (be motile).
SIM Media
culture used for endospore staining
bacillus cereus
culture media for endospore staining
Nutrient Agar (NA) slant
NA plate
Germination medium (GM)
stains used for endospore staining
malachite green
safranin
spore staining method used
Shaeffer-Fulton Spore Staining
- Grow B. cereus on NA slant. Incubate at 30-35 ͦC for 3-7 days.
- Prepare smear and fix by heat.
- Cover the smear with a piece of absorbent paper and flood with
__ ___
malachite green
Steam slide (under the fumehood) for 8-10 min such that evaporation
but not ___takes place. Keep smear saturated with malachite green.
Remove paper, cool slide, and wash thoroughly with tap water.
boiling
- Counterstain with __for 30-60 sec. Wash and dry.
safranin
Endospores are ___while vegetative cells are
___.
green
red
position of spore (3)
central
terminal
subterminal
shape of spore
ellipsoidal
oval
round
is an endospore that causes the bacterial cell to swell or bulge due to its size.
distended endospore
. Add 10 mL ___to a 36–48-hour old B. cereus culture grown on NA slant.
Dislodge the growth by scraping growth with a sterile wirelopp. Transfer
the suspension to sterile screw cap tube or dram vial no.8, add another
10 mL of GM and mix thoroughly.
GM
. Add 10 mL GM to a 36–48-hour old B. cereus culture grown on NA slant.
Dislodge the growth by scraping growth with a sterile wirelopp. Transfer
the suspension to sterile screw cap tube or dram vial no.8, add another
10 mL of GM and mix thoroughly.
what mehod
germination of bacterial spore
Determine the total cell and spore count by serially diluting the stock
suspension and pour plate on NA. Incubate plates as in ___
A
total cell and spore count is determined by ___
serial dilution of stock suspension and pour plate on NA
Place the remaining stock suspension prepared in step 1 in ___ oC water
bath and incubate for 10 min.
80oC
B label of GM of bacterial spore
placed in 80oC water bath and incubated for 10 mins, cool suspension and placed in pour plate on NA
what does the 80oC water bath do
kill vegetative cells while leaving heat-resistant spores intact
Allow the stock suspension (heated) to stand at room temperature for one hour.
why?
This incubation period allows the spores to germinate into vegetative cells.
how many heat treatment is done
2
Determine by counting colonies from the initial dilution and plating
what calculation
total cell and spores per mL in suspension
Determine by counting colonies from the heat-treated dilution and plating.
what calculation
b. Spores per mL in Suspension:
Calculate the difference between spores counted after the initial heat treatment and spores counted after the second heat treatment.
what calculation
c. Germinated Spores per mL in Suspension:
percent spores in original suspension formula
% spores in stock = B/A*100
% spores in stock = B/A*100
where
B =
A =
Where A is the count before heat treatment, and B is the count after heat treatment.
percent germinated stores formula
% germinated spores = B-C/B*100
% germinated spores = B-C/B*100
where
B - ?
C - ?
B - count after heat treatment
C - count after incubation and heat treatment
decolorizer for endospore staining
water
differentiateas between endospore forming bacteria and non endospore performing bacteria
endospore stain (differential stain)
protective, metabolically inactive structures
endospore
do endospore play a role in reproduction?
no
endospores are produced via
sporulation
a vegetative cell referred to as ___ produces the endospore within itself
sporangium
structure of endospore that makes it resistant to heat, radiation, chemical disinfection, and dessication
outer protein coat
genera of bacteria that causes endospores
bacillus
clostridium
present in soil, freshwater, and marine saprophytes
bacillus
known pathogen of bacillus genera
bacillus anthracis
causes anthrax
bacillus anthracis
a plant, fungus, or microorganism that lives on dead or decaying organic matter.
saprophytes
most are soil or aquatic saprophytes, some inhibit the human intestine
clostridium
causes tetanus
clostridium tetani
causes botulism
clostridium botulinum
causes gas gangrene
clostridium perfringens
causes pseudomembraneous colities
clostridium difficile
endospores would be observed as __ areas within the cells since crystal violet cannot stain it
clear
aqueous stain that does not bind strongly to cellular structures
malachite green
endospore form in the middle of sporangia
central endospore
endospore forming in the extreme end of sporangium
terminal endospores
endospore forming between the end and middle of cell
subterminal endospore
endospore can be shaped
spherical or elliptical
dormant structure that is resistant
to adverse conditions
endospore
bacterial culotures for Shaeffer fulton spore staining
b. cereus
e. coli
which did not germinate in 2nd plating and were able to
survive the 2nd heat treatment.
ungerminated spores
take note of formula for germination
+1
what other genera of bacteria produce spores?
Other genera of bacteria capable of producing spores include Sporolactobacillus, Sporosarcina, and Methylosinus. Sporolactobacillus forms spores that are less heat-resistant than Bacillus and are often found in soil and animal feed. Sporosarcina is a genus of cocci-shaped spore-formers involved in urea decomposition. Methylosinus produces exospores, which form externally and are also highly durable. These genera demonstrate diverse adaptations for surviving harsh environmental conditions.
how are the spores of actinomycetes differentiated from endospores of bacteria?
Actinomycetes and bacterial endospores have distinct differences as actinomycetes are considered exospores while bacterial spores are endospores. Additionally, actinomycetes spores are less heat resistant compared to bacterial endospores which can be attributed to their overall function where actinomycete spores are primarily for reproduction and dispersal while bacterial endospores are utilized as survival mechanisms under unfavorable conditions.
explain the physiological basis of an amyloid dye to detect the presence of spores. Is this method of spore detection applicable to fungal spores? why or why not?
Amyloid dyes, such as thioflavin T (ThT), are used to detect the presence of spores due to their affinity for amyloid or amyloid-like proteins found in the spore coat and exosporium. Here’s how it works:
Binding to Amyloid Proteins: Amyloid dyes have a high affinity for amyloid proteins, which are present in the outer layers of bacterial endospores. These proteins form a dense, protective barrier around the spore.
Fluorescence Enhancement: When amyloid dyes bind to these proteins, they undergo a conformational change that enhances their fluorescence. This results in a bright, easily detectable signal under fluorescence microscopy.
Selective Staining: The dye selectively accumulates in the amyloid-rich regions of the spore, providing a clear contrast between the spore and the surrounding vegetative cells or deb
Applicability to Fungal Spores
The use of amyloid dyes for spore detection is not universally applicable to fungal spores. Here’s why:
Presence of Amyloid Proteins: While some fungal spores contain amyloid or amyloid-like proteins, not all do. The presence and abundance of these proteins can vary significantly among different fungal species.
Staining Specificity: Amyloid dyes are highly specific to amyloid proteins. If fungal spores lack these proteins, the dye will not bind effectively, resulting in poor or no staining.
Alternative Staining Methods: Fungal spores are often detected using other staining methods, such as Melzer’s reagent, which reacts with starch-like compounds in the spores to produce a characteristic blue-black coloration
do endospores always germinate under favorable conditions? explain.
While endospores generally germinate under favorable conditions, there are some nuances to consider:
Presence of Germinants: Favorable conditions typically include the presence of specific nutrients (germinants) like amino acids, sugars, and nucleosides that can trigger germination. Without these germinants, even in a favorable environment, endospores may not germinate.
Temperature and pH: Endospores require an optimal temperature and pH range to germinate. If these conditions are not met, germination may not occur even if other factors are favorable.
Genetic Factors: Some bacteria have specific genetic controls that regulate the germination process. Mutations or variations in these genetic controls can affect the ability of endospores to germinate, even under favorable conditions.
Dormancy Period: Endospores can remain dormant for extended periods, sometimes even for decades. During this dormancy, they may not germinate immediately upon encountering favorable conditions. Instead, they might require a trigger or a specific signal to initiate the germination process
why are spore producing bacteria a problem in the food industry?
Heat Resistance: Spore-forming bacteria, such as Bacillus cereus and Clostridium botulinum, produce spores that can withstand high temperatures. This makes them difficult to eliminate through standard cooking and pasteurization processes.
Chemical Resistance: These spores are also resistant to many disinfectants and sanitizers used in food processing environments. This resistance allows them to survive cleaning procedures and contaminate food products.
Dormancy and Reactivation: Spores can remain dormant for extended periods, surviving in harsh conditions. When they encounter favorable conditions, they can germinate and multiply rapidly, leading to contamination and spoilage of food products.
Foodborne Illnesses: Spore-forming bacteria can produce toxins that cause severe foodborne illnesses. For example, Clostridium botulinum produces botulinum toxin, which can cause botulism, a potentially fatal illness.
Spoilage: In addition to causing illnesses, spore-forming bacteria can spoil food products, leading to economic losses for food producers and processors
test to determine whether bacteria are motile
motility test
motility test for agar is what percent
0.4%
detect bacterial growth (presence of bacteria) in motility test
Triphenyltetrazolium chloride (TTC)
when the TTC is __, it is going to be colorless
oxidized
reduced product of TTC
Formazan
reduced product of TTC that is colored ___
red
fuzzy apperance migrating away from the stab line means
positive
growth only along the stab line means
negative motility
refers to a specific type of bacterial movement where a large group of bacteria collectively migrate across a semi-solid surface, propelled by their flagella,
swarming
The ability of an organism to move by itself is called
motility
monotrichous organism
Vibrio cholerae
amphitrichous organism
Spirillium volutans
lopotrichous organism
Pseudomonas fluorescens)
peritrichous organism
e. coli
flagella consist of three parts
filament
hook
basal body
Composed of a protein called flagellin
filament
filament is composed of a protein called
flagellin
base of filament near cell wal
hook
Anchors filament & hook to cell wall
basal body
types of flagellar movement
run (counter clockwise)
tumble (clockwise)
straight line movement occurs when the flagella rotates
counterclockwise
run
turning the direction by clockwise movement of the flagella
tumbles
factors affecting flagellar synthesis
temperature
pH
presence of metallic ions
oxygen
nutrients
for flagellar synthesis, the temperature must be ___ than optimum for growth
5 degrees lower
pH for flagellar synthesis
closer to neutral
presence of ___ would block flagellin assembly by binding with its amino end
metallic ions
factor that accounts for active motility of aerobes and facultative
anaerobes
oxygen
high ___content reduces motility; no need to
move when there is an abundance in nutrients;
nutrient
___ inhibits
flagellar synthesis because it chelates poorly with metal ions at
normal pH
glucose
results from the random motion of the
water molecules bombarding the microbial cells and causing
them to move
brownian movement
example of microorganism exhibiting brownian movement
saccharomyces cerevisiae
staphylococcus aureus
independent movement brought by different
mechanisms for self-propulsion
true motility
examples of true motility for microorganisms
Bacillus megaterium, Escherichia coli
test to determine bacterial motility (2) for non pathogenic bacteria
Wet Mount
Hanging Drop
pathogenic bacteria motility testing
- Soft-agar stabbing (culture-based method)
visualization of flagella method
modified bailey’s method
Uses glass slides and
cover slips
wet mount
advantage of wet mount for motility testing
easier to
prepare
disadvantage of wet mount for motility testing
tend to
dry out quickly under the
heat of the microscope
light, thus, it is useful for
short-term observation
only
- Uses depression slides and cover slips
hanging drop technique
advantage for hanging drop
allows for longer-term observation and more reliable
observation of motility
disadvantage of hanging drop
more complex to prepare
disadvantages of hanging drop
specimen is
unstained, no contrast between specimen and background
solution for disadvantages of hanging drop and wet mount
use stains that will not kill the cells or distort the cells and
their structures, use phase contrast microscope
hanging drop method materials
cover glasses
depression slides
petroleum jelly
toothpicks
to increase contrast in hanging drop technique, move ___ all the way down and keep light as low as possible by closing __ ___
condenser
iris diaphragm
what part of the depression slide is observed
edge
does water current herding bacteria in the same direction a sign of motility?
no
- Uses wire needle, alcohol lamp, and motility medium* in test tubes
soft agar stabbing
Diffuse, hazy growths
that spread throughout the
medium rendering it slightly
opaque.
what result of soft agar stabbing
positive
Growth that is
confined to the stab-line, with
sharply defined margins and
leaving the surrounding
medium clearly transparent
what result of soft agar stabbing
negative growth
may be added
to facilitate the detection of motility
triphenyltetrazolium chloride
is a
redox indicator that is colorless in the oxidized
form but becomes an insoluble red precipitate
when reduced.
TTC
To determine presence/absence and arrangement of flagella
on bacterial cells
modified bailey’s stain
The ___would allow for higher affinity of the dye to
the flagella. The stain molecules will pile on the flagella, increasing its
thickness, therefore, making it easier to be viewed under the
microscope.
modrant
mordant component of bailey’s stain
binds to glycoproteins regardless of overall charge;
fixating agent
tannic acid
mordant component of bailey’s stain
colors the cell and the flagella red
basic fuschin
binds with Tannic Acid to form mordant
FeCl3 in 6 H20
fixating agent for Basic Fuchsin
formalin
– functions for hydrolysis, prevent tannin-iron
reaction that can color the cell with dark gray instead of red
concentrated HCl
- commonly used for staining Mycobacteria; with
high affinity for mycolic acids
phenol + basic fuschin
Phenol + Basic Fuchsin is the component of
Ziehl’s carbol fuschin
Ziehl’s carbol fuschin components
Phenol + Basic Fuchsin
is a stain specifically used to identify acid-fast bacteria, like Mycobacterium tuberculosis, which causes tuberculosis, under a microscope; it allows for the visualization of these bacteria due to their unique cell wall composition that resists decolorization with acid alcohol, making them appear bright red against a counterstained background.
Ziehl carbol fuschin
another difficulty in flagellar examination is the
ease with which bacteria shed these delicate appendages unless the cultures are properly handled
to prevent shedding of flagella, ___ are utilized
specially cleaned slides
specially prepared smears
bacterial cultures for motility medium
proteus vulgaris
staphylococcus aureus
culture media utilized for motility testing
NA slant
Motility medium tube
stain used for motility testing
modified Bailey’s flagellar stains
ziehl’s carbol fuschin
(The meniscus
of the syneresis should ___touch the bottom of the depression.
not
different method for observation of motility band
- With a pipette, get one drop of bacterial suspension prepared in part A and carefully add to MM. Incubate as above.
- To another tube of MM, inoculate bacterial suspension by stabbing with a wire
needle. Incubate as above.
modified bailey’s method
- If possible, use new slides which are devoid of ___.
grease
modified bailey’s method
Soak in a ___ cleaning fluid, wash in water, and rinse in 95% ethyl
alcohol; then wipe with a clean piece of cheesecloth.
dichromate
- Pass each slide back and forth through a __ for some time or until the appearance of an ___color in the flame. Cool slides gradually to prevent
breakage.
flame
orange
Flood smear and keep saturated with freshly filtered ___ A (filtering directly to
slide is best) and allow to act for 3.5 min without heating. (A powdered
commercial form of this stain is now available). Handle slides with forceps or
relhr clothes pin.
mordant
To minimize the
effects of stain precipitates and other artifacts, only use slides and ___.
degreased
extremely fragile. Handle the preparations very gently to minimize damage
bacterial flagella
1.Why should the temperature be 5 °C lower than optimum for growth in incubating cultures for the study of flagella?
Incubating cultures at a temperature 5°C lower than its optimal growth temperature can slow overall cellular growth while preserving the structural integrity and functionality of flagella. This approach minimizes the denaturation of flagellar proteins, such as flagellin, and reduces stress on the basal body and intraflagellar transport systems, which are sensitive to temperature fluctuations. Additionally, slower growth rates allow for more precise observation of flagellar assembly and function without interference from rapid cell division or metabolic activity. This controlled environment is crucial for studying the dynamics of flagella in detail.
- What is the function of EDTA in the motility medium?
Ethylenediaminetetraacetic acid (EDTA) is utilized in the motility medium to prevent aggregation (clumping of cells), enhance motility, and stabilize the medium. EDTA reduces the clumping of cells which can help observe true motility, disrupt the cell membrane to make it easier for motile bacteria to move, and maintain the consistency and clarity of the medium to ensure accurate results.
- Why are smears for flagella staining not fixed by heat?
Heat fixing is not suitable for preparing smears for flagella because of the fragility of bacterial flagella, the potential for cell distortion, and the preservation of morphology. These structures can be damaged or destroyed by heat fixing, causing them to be invisible or deformed under the microscope. It can also cause bacterial cells to burst or shrink, producing artifacts that could be misinterpreted for flagella.
- Why do you have to use freshly prepared flagellar stains?
Using freshly prepared flagellar stains is important because they ensure optimal staining quality and clarity. Stains that are not freshly made may lose potency or become contaminated, resulting in poor contrast and visibility of flagella. Fresh stains provide consistent results, allowing for accurate observation and analysis of the flagellar structures in microorganisms.
- Can bacteria have flagella but are not motile? Explain.
Yes, bacteria can still move even without possessing a flagella and it could be possible due to several reasons. (1) Environmental factors like viscosity of the habitat causing the organism to have difficulties in moving from one space to another or rather immobile. (2) Physiological factors like the stage of the species’ life, some species can only manifest their flagellas at a certain time of their lifetime (Zhuang et al 2019). In addition, some species only use flagellas on attaching to surfaces, and not be motile (PNAS 2013).
. total vegetative cells and spores per of ml of stock formula
𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑛𝑜.𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑝𝑒𝑟 𝑝𝑙𝑎𝑡𝑒 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟/
𝑣𝑜𝑙𝑢𝑚𝑒 𝑝𝑙𝑎𝑡𝑒𝑑 𝑖𝑛 𝑚l
Spores/mL in suspension formula
= 2nd plating + 3rd plating
Germinated spores/mL of suspension formula
2𝑛𝑑 𝑝𝑙𝑎𝑡𝑖𝑛𝑔/
2𝑛𝑑 𝑝𝑙𝑎𝑡𝑖𝑛𝑔+3𝑟𝑑 𝑝𝑙𝑎𝑡𝑖ng
% spores in stock formula
2𝑛𝑑 𝑝𝑙𝑎𝑡𝑖𝑛𝑔+3𝑟𝑑 𝑝𝑙𝑎𝑡𝑖𝑛𝑔/
1𝑠𝑡 𝑝𝑙𝑎𝑡𝑖𝑛𝑔 𝑥 100
% germinated spores in stock
2𝑛𝑑 𝑝𝑙𝑎𝑡𝑖𝑛𝑔/
2𝑛𝑑 𝑝𝑙𝑎𝑡𝑖𝑛𝑔+3𝑟𝑑 𝑝𝑙𝑎𝑡𝑖𝑛𝑔 𝑥 100
