Bacterial Morphology Part 1 Flashcards
is an intricate structure made up of different layers that have to fulfill several essential and crucial functions of the cell.
bacterial cell wall
aside from protectively shielding the cell against a hostile environment, what is the function of bacterial cell wall
allow communication with its surroundings to find optimal conditions for growth and development of the bacterial cell
characteristic of bacterial cell wall that maintains the shape of the bacterium and enables the transport of large molecules into and out of the cell.
rigidity
The structure and composition of different cell walls can vary considerably depending on the ___ and thus exhibit differential reaction to different reagents and staining procedures
organism
The Gram stain was developed in 1884 by a Danish scientist and physician named
Hans Christian Joachim Gram
The Gram stain was developed in ___ by a Danish scientist and physician, Hans Christian Joachim Gram.
1884
It is one of the most useful and widely used differential staining procedures employed in bacteriology
gram staining
gram staining divide bacteria into two broad groups
gram positive
gram negative
The difference in staining is due to the differences in the surface layer composition of the two cell types resulting in differential capability to retain the purple color of ___ ___ during decolorization with alcohol.
crystal violet
Gram ____bacteria are decolorized by the alcohol, losing the purple color of crystal violet
negative
Gram negative bacteria are decolorized by the ___, losing the purple color of crystal violet
alcohol
Gram ____ bacteria are not decolorized and remain purple.
positive
After decolorization, ___, a red counterstain, is used to impart a red/pink color to the decolorized Gram negative organisms
safranin
Additionally, Gram-stained cells are easily viewed by ___microscopy where cell shape, size and arrangement can be easily determined.
light
Staining techniques often require fixing specimens on the slide (called a
smear
Staining techniques often require fixing specimens on the slide (called a smear) to immobilize and kill the microbes with just enough ____to preserve the integrity of their cellular components for observation.
heat
are composed of polysaccharides but may also contain polyalcohol and polyamines.
capsules
Capsules are composed of ___but may also contain polyalcohol and polyamines.
polysaccharides
Capsules are composed of polysaccharides but may also contain __ and ___.
polyalcohol
polyamines
They are external to the cell wall for Gram positive or to the outer membrane for Gram negative bacteria
capsules
capsules are external to the __if gram positive
cell wall
capsules are external to the ___ if gram negative
outer membrane
When the material is tightly bound and remains attached to cells and there is a defined shape, it is referred to as
capsule
when the capsule is loosely bound and there is no distinct boundary, it is called a
slime layer or glycocalyx
capsule allows for cell __ to surfaces and protect cells from dessication, chemical and physical inhibitory factors as they contribute to biofilm formation
adhesion
They can boost the bacterium’s virulence, protecting them from the host’s immune system and confer to the cells antigenic determinants leading to variation in pathogenicity.
capsule
here are some ___developed based on capsule antigenicity
vaccine
Capsules/slime layers can be visualized through
electron microscopy
phase contrst microscopy
will provide a colored background as the large particles of ink will neither penetrate the tight layers of the capsule nor stain the bacterium, thus, allowing visualization of cells and capsules.
negative staining
Other methods are based on the use of a ___ dye that interacts with the negative ions of the bacterial cell and an ___stain used to color the background.
basic
acidic
he capsules will then be revealed as a clear ___between the colored background and the stained cel
halo
Some procedures utilize mordants like ___, ___, and ___ which Precipitate the capsular material making the capsules visible
metal ions
alcohol
acetic acid
are concentrated deposits of substances in certain bacterial cells.
granules or inclusion bodies
function of granules or inclusion bodies
as energy reserves and as reservoirs of structural building blocks
granules or inclusion bodies are usually enclosed by a ___ membranes that partition them off in the cell and stain more deeply than the remainder of the cell, thus can be seen with the light microscope
single layer
The presence of such inclusion bodies confers an additional advantage to the cell as it reduces the osmotic stress when the same amount of the substance is dissolved in the cytoplasm.
granules or inclusion bodies
granules or includion bodies’ presence confers an additional advantage to the cell as it reduces the ___ ___when the same amount of the substance is dissolved in the cytoplasm.
osmotic stress
One of the carbon storage granules consists of lipids which include
polyhydroxyalkanoate (PHA) class of carbon and energy storage polymers
These are synthesized by cells when there is an excess of carbon and are utilized for biosynthetic or energy purposes when needed.
polyhydroxyalkanoate (PHA) class of carbon and energy storage polymers
a lipid that is formed from - hydroxybutyric acid units.
polyhydroxybutyric acid (PHB)
Various proteins are known to associate with these granules; thus, their surfaces can be functionalized to create biotechnologically applicable biobeads which can replace existing colloidal systems for protein purification, drug delivery and enzyme immobilization
polyhydroxybutyric acid (PHB)
Various proteins are known to associate with these granules; thus, their surfaces can be functionalized to create biotechnologically applicable ___which can replace existing colloidal systems for protein purification, drug delivery and enzyme immobilization
biobeads
They usually occur in the cytoplasm but recently PHB was reported to be associated with the
nucleoid
Another storage product, which also serves as storage for both carbon and energy is
glycogen
polymer of glucose
glycogen
It is produced when there is an excess of carbon in the environment and is utilized when carbon is limited.
glycogen granules
Glycogen differs from ___in the way in which the glucose units are linked together
starch
Many microorganisms accumulate inorganic phosphate (P04) as ___
polyphosphate
Many microorganisms accumulate inorganic phosphate (P04) as polyphosphate or as ___ in the cell in the form of granules when there is excess of phosphate or a sulfur source
elemental sulfur
e phosphate granules can be degraded and used as sources of phosphate for what biosyntheses
nucleic acid
phospholipid
The phosphate granules can be degraded and used as sources of phosphate for nucleic acid and phospholipid biosyntheses and in some cases can be used to make the energy-rich compound
ATP
Bacterial cells can oxidize/reduce sulfur during energy metabolism called
chemolitotrophy
Bacterial cells can oxidize/reduce sulfur during energy metabolism (chemolithotrophy) or CO2 fixation called ___
autotrophy
They are called ___granules because of the capacity to be stained in a different shade from the different components of the cell.
metachromatic
bacterial cultures used in gram staining
Bacillus megaterium
Escherichia coli
Bacillus subtilis
culture medium for gram staining bacteria
NA slant
regeants and stains used for cell wall staining
gram’s staining set
3% KOH
Streak
for 16-22
E. coli, B. megaterium and the unknown bacterium on NA. Incubate at ___ oC for 16-22 hours
30
Stain with crystal violet for ___min and rinse thoroughly but gently with tap water.
1
Cover smear with Gram’s iodine for __ min. Wash with tap water. Drain excess
moisture and dry.
1-2
Decolorize by dripping 95% ethanol over tilted slide for ___ sec. Wash
immediately with water.
10-15
Decolorize by dripping ___ over tilted slide for 10-15 sec. Wash
immediately with water.
95% ethanol
Counterstain with safranin for ___sec. Wash with water. Drain excess moisture
and dry.
45
b. subtilis gram reaction
gram positive (purple)
e. coli gram reaction
gram negative
Method of determining gram reaction using KOH
Gregersen’s method
- Deposit 1-2 drops of freshly prepared ____ on a glass slide.
3% KOH
- Deposit ___drops of freshly prepared 3% KOH on a glass slide.
1-2
Gregersen’s method of determining gram reaction is also called
Potassium Hydroxide test
The cell walls of Gram-___bacteria have a thin layer of peptidoglycan. When exposed to 3% KOH, this thin layer is easily dissolved, causing the cell walls to break down. This leads to cell lysis and the release of cellular contents, including DNA. The released DNA makes the mixture viscous and stringy, which is a positive result for the KOH test
negative
In contrast, Gram-___bacteria have a thicker peptidoglycan layer in their cell walls. This thicker layer is resistant to the action of KOH, so the cells do not lyse, and no DNA is released.
positive
the higher the ___, it gives a false Gram-negative.
pH
The higher pH can affect the stability of the ___layer in Gram-positive bacteria.
peptidoglycan
- Can you vary the primary stain and counterstain in the Gram staining procedure? Explain
Primary stains and counterstain in Gram staining procedure may vary because the primary stain needs to enter all cells, while the counterstain only needs to color the cells that don’t retain the primary stain after decolorization. Crystal violet and gentian violet can effectively act as the primary stain, targeting the peptidoglycan in cell walls. Likewise, safranin and basic fuchsin counterstains by providing contrasting colors to visualize Gram-negative bacteria.
alternative for primary stain of crystal violet
gentian violet
aleternative for safranin
fuchsin counterstain
- What would be the effect of replacing iodine with other oxidizing agents?
Replacing iodine with another oxidizing agent in Gram staining will likely result in the loss of differentiation between Gram-positive and Gram-negative bacteria, as another oxidizing agent might not efficiently “fix” the crystal violet dye into the peptidoglycan layer of the Gram-positive bacteria’s cell wall.
In Gram staining, iodine acts as a mordant, forming a complex with the crystal violet stain to help retain the crystal violet. If this complex is replaced, it may be compromised, resulting in inaccurate staining and visualization of the bacteria.
- What is the importance of using freshly prepared KOH?
Potassium Hydroxide (KOH) is crucial for accurate differentiation between Gram-positive and Gram-negative bacteria due to its chemical properties and interaction with bacterial cell walls. Degradation over time may result in inaccurate findings in tests that rely on KOH’s specific alkalinity. A fresh KOH solution provides rapid and reliable disruption, releasing DNA and making the solution viscous and “stringy.” Inaccurate results can lead to bacterial misidentification, affecting clinical and research settings.
KOH is a strong base and can absorb carbon dioxide (CO₂) from the air over time, forming potassium carbonate (K₂CO₃). This reaction can reduce the effectiveness and concentration of the KOH solution. Freshly prepared KOH ensures that the solution is at its full strength and reactivity.
- What is the influence of pH and age of the cultures on Gram reaction?
The pH and age of cultures can affect Gram staining results. Acidic or extreme pH conditions can damage the bacterial cell wall, causing Gram-positive bacteria to appear Gram-negative. Similarly, older cultures (over 24 hours) may lose their ability to retain the crystal violet dye due to cell wall degradation, leading to Gram-variable or Gram-negative results. For accurate staining, use fresh cultures (18-24 hours old) and maintain neutral pH conditions. Observed in this experiment with Bacillus megaterium, it gave a false Gram-negative by staining its cell wall with pink.
- What is the significance of using known/reference cultures in Gram staining?
The utilization of known or reference cultures in Gram staining is important for two primary reasons. First, it minimizes corrections during investigation of the culture/organism, as pre-existing information eliminates the need to determine its behavior. Second, known cultures serve as a reference point for understanding other cultures that exhibit similar characteristics and behavior (Smith and Hussey 2005). The use of known cultures creates an assurance that quality control is met, ensuring that the staining technique is properly executed and optimize results.
bacterial samples for bacterial capsule staining
Acetobacter aceti var. xylinum
Bacillus cereus
negative control for capsule since it does not produce one
Citrobacter freundii
culture media used (2) in staining capsules
Nutrient Agar + 2.0% Glucose (NAG) slant
Nutrient Agar + 2.0% Sucrose (NAS) slant
duguid’s method is also known as
negative staining technique
The principle of Duguid’s method is based on the use of an acidic dye, such as
india ink
nigrosin
Since the surface of most bacterial cells is ___charged, the acidic dye is repelled by the cell surface, resulting in a clear, unstained area around the cells.
negatively
This clear area represents the bacterial capsule, which appears as a ___around the cells against a dark background
halo
do you heat duguid’s method?
no
why is heat fixing not used in negative staining for capsule?
he primary goal of negative staining is to observe bacterial capsules, which are delicate and can be easily distorted or destroyed by heat. Heating can cause the capsules to shrink or become misshapen, making it difficult to accurately observe and interpret their presence and structure.
. From the same cultures in A, emulsify a small amount of the bacterial growth in a loopful of 6% ___solution at opposite ends of the slide.
Anthony’s method
glucose
anthony’s method
the culture is stained using
crystal violet
after staining with CV, the specimen is washed with ____
Anthony’s method
copper sulfate solution
Examine under OIO. Capsules should appear __ ___ or colorless, while cells are ___ ___.
Anthony’s method
light blue
dark purple
uses a combination of acidic and basic dyes to stain the background and bacterial cells, respectively
anthony’s method
The capsule, being ___, does not take up the stain and appears as a clear halo around the stained bacterial cell against a colored background.
non-ionic
percent of copper sulfate solution
20%
crystal violet solution percent
1%
acts as a decolorizing agent, modrant, and gentler than water in Anthony’s method
copper sulfate
dye used in Maneval’s staining method
congo red
maneval’s stain
combines both negative and positive staining techniques. The background is stained with an acidic dye, while the bacterial cells are stained with a basic dye
Maneval’s method
Using a sterile loop, mix a small amount of bacterial culture with the __ ___drop and spread it across the slide to create a thin smear.
Maneval’s method
congo red
Flood the smear with Maneval’s stain and let it react for __ minutes.
maneval’s method
4
Bacterial cells will appear pink or red, while the capsules will be visible as clear halos around the cells against a blue background.
what staining for capsule
Maneval’s method
Maneval’s method is particularly useful for visualizing capsules of bacteria such as
Klebsiella pneumoniae, Azotobacter spp., and Rhizobium spp
Exhibits high specificity for amyloid fibrils due to hydrogen bonding and hydrophobic interactions with β-pleated sheet structures
congo red
composition of maneval’s stain
acid fuschin, phenol, ferric chloride, acetic acid
is an acidic dye that stains the background but not the bacterial cells or their capsules in Maneval’s method
congo red
Congo Red creates an ___environment on the slide, which is necessary for the subsequent staining with Maneval’s stain.
acidic
acid fuchsin is a ___ dye
acidic
this acidic dye binds to the background, leaving the bacterial cells and capsules clear.
Maneval’s stain
acid fuschin
This component changes the color of the Congo Red background from red to blue, creating a high-contrast background for the stained cells.
what component of Maneval’s stain
acetic acid
final result in maneval’s stain
The bacterial cells appear __ or ___due to the uptake of acid fuchsin.
pink
red
final result in maneval’s stain
The background, initially stained red by Congo Red, turns __after the application of Maneval’s stain, providing a clear contrast.
blue
Typically forms large, opaque colonies due to its ability to ferment glucose.
Colonies appear to have a moist and smooth texture.
Being Gram-positive, rod-shaped, and can produce endospores, they contribute to its resilience in various environments.
what media of B. cereus
NAG slant
Similar to NAG slant colony morphology.
Can exhibit a more pronounced acid production due to its sucrose fermentation.
Colonies may appear more translucent and have a slightly different texture compared to those on NAG slants.
what media of B. cereus
NAS slant
Typically forms smaller, translucent colonies with a mucoid texture due to its ability to produce cellulose.
Being Gram-negative and rod-shaped, it can also live in aerobic-conditioned environments.
what media of Acetobacter aceti var xylinum
NAG slant
Shows enhanced growth and more pronounced mucoid texture as it utilizes sucrose for energy.
Colonies appear more viscous due to increased cellulose production.
what media of Acetobacter aceti var xylinum
NAS slant
why are smears for capsule staining not fixed by heat?
Smears for capsule staining are not heat-fixed because the heat can distort or destroy the capsule. Since most capsules are over 95% water, heat fixation can dehydrate the capsule, leading to false negatives. Heat can also cause the bacterial cell to shrink, resulting in a clear zone around the cell, which may be mistaken for a capsule and lead to false positives. Therefore, capsule staining methods usually depend on revealing the capsule indirectly by staining the background and the cells, leaving the capsule as a clear halo.
which of the two media was more favorable for capsule formation of acetobacter aceti var. xylinum and bacillius subtilis. explain.
For Gluconacetobacter xylinus (formerly Acetobacter aceti var. xylinum), the Nutrient Agar + 2.0% Glucose (NAG) slant is generally more favorable for capsule formation. This is because glucose is a simpler sugar readily utilized by the bacteria to produce cellulose, a key capsule component.
Bacillus subtilis, on the other hand, can utilize both carbon sources but exhibits a slight preference for the Nutrient Agar + 2.0% Sucrose (NAS) slant due to its evolved specific metabolic pathways. As a disaccharide, sucrose provides a more complex carbon source to the bacterium, which enhances the production of extracellular polysaccharides essential for capsule formation. Additionally, sucrose plays a role in surface motility and root colonization for B. subtilis.
what would be the effect of prolonged incubation on capsule formation
Prolonged incubation can have substantial effects on capsule formation, growth phases, and nutrient depletion. Capsule production may vary, leading to irregular or reduced production in some species. Nutrient deficiency can also impair capsule formation. Over time, capsules may degrade due to bacterial enzymes or environmental factors, making it difficult to determine their presence or size. Additionally, phenotypic changes, such as variations in capsule expression, may occur, and some bacteria may lose their ability to produce capsules. Environmental factors, including nutrient availability and incubation temperature, significantly influence capsule development.
explain the principleso f anthony and maneval capsule staining procedures
The principles of the Anthony and Maneval capsule staining procedures is used to visualize bacterial capsules, which are primarily protected by layers surrounding bacteria. Each method employs different staining principles and reagents such as:
Anthony’s capsule Staining Procedure – this technique uses crystal violet to stain bacterial cells, while the usage of copper sulfate acts as a mordant and decolorizer. The capsule remains unstained, appearing to have a clear or whitish halo around the purple-stained cells against a dark background.
Maneval’s capsule Staining Procedure – although not performed in this experiment, this method combines negative staining with Congo red for the background and acid fuchsin in Maneval’s solution to stain bacterial cells. Though the capsule is not stained, it appears as a clear halo between the colored cell and the background.
give the various advantages of capsulated bacteria over non-capsulated ones
Capsulated bacteria possess more advantages than non-capsulated bacteria, primarily due to survivability and virulence properties. The capsule acts as a barrier that prevents processes like phagocytosis (immune cells), thus preventing destruction. This layer also retains moisture, preventing desiccation and enabling survival in harsh environments. The chemical complexity of capsulation can provide adherence to the host (organisms, tissues, etc.) for colonization and infection. These capsules also mask the bacteria from any immune response from its host through surface antigens to prevent recognition. Above all, these characteristics are often labeled as increased virulence hence capsulation is a variable for disease-causing bacteria.
bacterial cultures for bacterial storage granules
bacillus megaterium
bacillus cereus
culture medium for bacterial storage granules
N-limited Glucose Yeast Extract Agar (GYEA) slant
N-limited GYEA slant has what effect on the cells
Nitrogen limitation encourages the accumulation of storage materials like lipids within the cells.
is a lipid-soluble dye that specifically stains lipid inclusions within the cells. Flooding the smear with this dye for 15 minutes allows the dye to penetrate the cells and bind to the lipids.
sudan black
Excess Sudan Black B stain is drained off without adding ___. This step prevents the loss of stain from the lipid inclusions.
water
is used to rinse the slide under a fume hood as it acts as a clearing agent, removing any excess Sudan Black B stain that is not bound to lipids.
xylene
is a basic dye that stains the bacterial cells pale red, providing a contrasting background to the darkly stained lipids.
safranin
Intracellular lipids will be stained __ to ___, appearing as distinct granules within the bacterial cells.
blue to black
is a solution containing iodine (I₂) and potassium iodide (KI). When mixed with the bacterial culture, the iodine in the solution interacts with starch and glycogen granules present within the cells.
lugol’s iodine
are composed of amylose and amylopectin. When iodine molecules come into contact with amylose (the linear component of starch), they fit into the helical structure of the amylose chains, resulting in a dark blue or purple color.
starch granules
Starch granules are composed of amylose and amylopectin. When iodine molecules come into contact with amylose (the linear component of starch), they fit into the helical structure of the amylose chains, resulting in what color
dark blue or purple
is a highly branched polysaccharide, when reacting with iodine, hey bind to the polysaccharide chains, resulting in a reddish-brown or mahogany color.
glycogen
Glycogen is a highly branched polysaccharide. When iodine molecules interact with glycogen, they bind to the polysaccharide chains, resulting in a
reddish brown or mahogany
binds to acidic components within the cells, particularly nucleic acids and metachromatic granules
methylene blue
are storage forms of inorganic phosphates. They have unique staining properties, causing them to stain differently than the rest of the cell.
metachromatic granules
metachromatic granules are also known as
volutin granules
metachromatic granulesa ppear __ or ___due to the metachromatic effect (a change in color due to the chemical nature of the granules).
blue or purple
The rest of the cells will be stained l___ ___ providing a contrasting background for the darker metachromatic granules.
light blue
give examples of other granules that can be found among prokaryotes
Prokaryotes utilize storage granules to store nutrients and reserves like lipids, polyphosphate, sulfur, or nitrogen. Polyphosphate granules store phosphate ions for cellular processes and can sequester toxic heavy metal ions. Sulfur granules, commonly found in bacteria that use hydrogen sulfide, store elemental sulfur. Some bacteria also form granules that contain lipids, iron, and nitrogen, allowing them to adapt to different environmental conditions.
under favorable conditions, will all bacteria produce at least one type of storage granule? cite specific examples.
No, not all bacteria will produce storage granules under favorable conditions. The production of storage granules depends on the metabolic capabilities and environmental adaptations of each bacterial species. For instance, E. coli can produce polyhydroxy butyrate (PHB) granules when there is an excess of carbon sources but a limitation of other nutrients such as nitrogen. Bacillus spp., on the other hand, is known to have polyphosphate granules as phosphate reserves in energy storage and regulation. Moreover, the production of storage granules varies based on the environmental context such as Cyanobacteria storing glycogen granules as a carbohydrate reserve as well as Mycobacterium spp. producing lipid inclusions that are utilized under stress conditions.
do all bacteria contain storage granules at all stages in their life. explain
Not all bacteria have storage granules at every stage of their life cycle. These granules are high-molecular-weight polymers formed from excess nutrients in bacteria. Their abundance and composition vary with species, nutritional availability, and life cycle stage. Storage granules operate as reserves, allowing bacteria to survive during situations of nutrient scarcity. The composition depends on the type of excess nutrient, such as glycogen, poly-β-hydroxybutyrate, or polyphosphate. Environmental factors also affect the prevalence and activity of certain microorganisms. Aerobic granules, thick biofilms used in wastewater treatment, go through numerous developmental phases, each with a shift in dominant microorganisms. The presence and composition of storage granules in these biofilms differ depending on their stage of development and nutrition availability. In addition, nutrient levels and bacterial physiological status impact biofilm formation and dispersal.
in this exercise, what will be the outcome if B. megaterium was grown in a rich medium?
When Bacillus megaterium is grown in a rich medium, it accumulates large amounts of storage granules, particularly polyhydroxy butyrate (PHB), as a carbon reserve. The abundant nutrients also contribute to promoting rapid growth, higher biomass production, and the synthesis of beneficial metabolites like butanediol and fatty acids. In addition, the bacterium mas exhibit physiological adaptations, like spore formation and biofilm production, which can enhance its survival and industrial utility.
discuss the basis of at least one of the staining procedures
Metachromatic staining. The basis of this staining relies on the phenomenon of metachromasia, this refers to the ability of certain dyes to change color when they bind to polymerized polyanions, such as the polyphosphate in volutin granules.
A known procedure that uses this particular staining technique is the selective identification of the species Corynebacterium diphtheriae, the causative agent of diphtheria. Assuming positive results, the metachromatic granules appear dark purple or black and the cytoplasm is pale green. These are key characteristics in identifying C. diphtheriae (Meredith and Ulrich 2012) .
look at the results in the ppt and online
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