Cell Staining Flashcards
What colour do gram positive bacteria stain?
Purple
What colour do gram positive bacteria stain?
Pink
Steps in gram staining
- Crystal violet (primary stain)
- Gram’s iodine (mordant)
- Alcohol (decolourizer)
- Safranin (counterstain)
List potential mistakes that can be made in gram staining
How will you identify that these mistakes occurred?
- Clear slide
- - Not heat fixing slide - Not enough decolourization
- - Gram -ve bacteria appear purple and pink - Clear slide
- - Overheating/not letting slide completely dry - Too much decolourization
- - Gram +ve bacteria will appear purple + pink - Forgetting to counterstain
- - Purple gram +ve or clear gram -ve
Note: If culture is not pure then purple and pink can be seen
Match terms with descriptions Cocci Diplococci Streptococci Staphylococci Spirilla Cocci Rod shaped Vibrios Spirilla Sarcina
Rigid spiral shaped cells
Roughly spherical cells
arise when cocci divide and remain together to form pairs
Comma shaped bacteria
Grape like clusters
Forms group of 8 cells
Many have tufts of flagella at one or both ends.
may occur singly, but some remain together to form pairs or chains
Can occur singly but also in groups
when cells adhere after repeated divisions in one plane.- chains
Cocci - Roughly spherical cells
Diplococci - arise when cocci divide and remain together to form pairs
Streptococci - when cells adhere after repeated divisions in one plane - chains
Staphylococci - Grape like clusters
Spirilla - Rigid spiral shaped cells
Cocci - Can occur singly but also in groups
Rod shaped - may occur singly, but some remain together to form pairs or chains
Vibrios - Comma shaped bacteria
Spirilla - Many have tufts of flagella at one or both ends.
Sarcina - Forms group of 8 cells
Examples of Diplococci
Streptococcus pneumonia, Moraxella catarrhalis, Enterococcus spp, Neisseria gonorrhea.
Examples of bacteria with Tetrad (group of 4 cells) arrangement
Aerococcus, Pediococcus, and Tetragenococcus
Examples of Sarcina
Sarcina aurantiaca, Sarcina lutea, Sarcina ventriculi
Examples of Streptococci
Streptococcus pyogenes, Streptococcus pneumonia, Streptococcus mutans
Examples of Staphylococci
Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus aureus, Staphylococcus capitis.
Examples of Bacilli
Salmonella enterica and Bacillus cereus
Example of Diplobacilli
Moraxella bovis
Examples of Streptobacilli (chains)
Streptobacillus moniliformis, Streptobacillus Levaditi
Examples of coccobacilli
resemble cocci and bacilli
Chlamydia trachomatis, Haemophilus influenza, Gardnerella vaginalis.
Example of Palisade (picket fence shape) bacterial cell
Corynebacterium diphtheria
Examples of Vibrio
Vibrio parahaemolyticus, Vibrio cholera
Spirochetes - characteristics
Helical shape
Flexible and have an axial filament
Filaments run throughout the length of the bacteria
Filaments help in twisting the motion of the bacteria
Spirochetes
Leptospiraspecies (Leptospira interrogans), Treponema pallidum, Borrelia recurrentis
Spirilla
- Similar in structure with spirochetes but are more rigid
- They, too, have a flagellum but lack the endoflagella like in spirochetes.
Campylobacter jejuni, Helicobacter pylori,
Appendaged bacteria - characteristics
- Unique structure like pillus or fimbriae
- More virulent than other bacteria that do not form these appendages.
Examples of appendaged bacteria
Neisseria gonorrheae, the agent of Gonorrhea.
Box shaped bacteria - characteristics and examples
- resemble a box.
Haloarcula marismortui.
Club shaped rod bacteria - characteristics and examples
- thinner on one side than the other.
Corynebacterium.
Filamentous bacteria - characteristics and examples
- Long, thin, and filament-shaped.
- They, sometimes, divide to form branches resembling strands of hair or spaghetti called mycelium.
Actinomycetes.
Triangular shaped bacteria - characteristics and examples
- Triangular in shape.
Haloarcula.
Pleomorphic bacteria - characteristics and examples
- Do not have a specified shape are included in this group.
- Can change their shape, but in pure culture, they appear to have a definite form.
Mycoplasma pneumoniae, M. genitalium.
Stalked bacteria
bacteria that possess a stalk on one end of the cell.
Examples: Caulobacter crescentus.
Star shaped bacteria
- look like stars
Stella humosa.
Mordant - function in gram stain
Gram’s iodine forms a complex with crystal violet in the stain that has attached more tightly to the cell wall of gram positive bacteria than that of the gram negative bacteria.
Gram +ve vs Gram -ve cell wall rxns in gram stain
- gram positive bacteria stain violet as a result of the presence of a thick peptidoglycan layer in the walls of their cell, the gram negative bacteria stain red,
- due to the thinner peptidoglycan layer in their cell wall (a thicker peptidoglycan layer allows for the retention of the stain, but a thinner layer does not).
What causes the different stain colours to occur in gram +ve and gram -ve bacteria?
- Differences in the thickness of the peptidoglycan layer on the cell walls Gram positive bacteria will retain the crystal violet stain after the de-colorization
Decolourizer - function in gram stain and effect on gram +ve and gram -ve
Achieves its purpose by dehydrating the peptidoglycan layer by tightening and shrinking it.
In doing so, large crystal violet cannot penetrate the tightened layer of peptidoglycan, and hence it is trapped in the cell wall of gram positive bacteria.
On the other hand, the outer membrane of the gram negative cells cannot retain the crystal violet iodine complex and hence the color is lost.
Why doesn’t Safranin affect gram +ve cell colour?
Safranin is a lighter stain as compared to crystal violet and thus it does disrupt the purple coloration in the gram positive cells.
Crystal violet - theory - gram stain
In an aqueous solution, crystal violet dissociates into ions of CV+ and CV-. These ions penetrate the walls and membranes of both gram positive and negative cells.
CV+ will interact with the negatively charged components of the bacterial cells, and take up the purple coloration.
On adding iodine, iodine cations (I- or I3-) interact with CV+, which results in the formation of larger complexes of CVI within the cytoplasm and the outer layers of the cell.
Decolourization - theory
This results in the loss of the outer membrane, which in turn leaves the peptidoglycan layer exposed.
Gram +ve
Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell wall and prevents the stain from exiting the cell.
Gram -ve
Cell walls of gram negative organisms do not retain this complex when decolorized.
Peptidoglycans are present in the cell walls of gram negative organisms, but they only comprise 10-20% of the cell wall. Gram negative cells also have an outer layer which gram positive organisms do not have; this layer is made up of lipids, polysaccharides, and proteins. Exposing gram negative cells to the decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells.
For the gram negative cells, ethanol causes the walls to be leaky and hence they cannot hold the large complexes of CV-L during de-colorization.
There can be a variation of pink and purple gram stain for certain bacteria
- What can cause this other than gram stain errors?
Arthrobacter, Actinomyces and Corynebacterium - have a cell wall that is particularly sensitive to breakage during cell division.
This results in gram negative staining of the gram positive cells.
Cultures of Clostridium and Bacillus, the reduced thickness of peptidoglycan during growth coincides with an increased number of cells that in turn stain gram negative.
____-__________ may result in the identification of false gram-negative results, whereas ______-___________ may result in the identification of false gram-positive results.
Over-decolorization may result in the identification of false gram-negative results, whereas under-decolorization may result in the identification of false gram-positive results.
Smears that are too _____ may retain too much ______ stain, making the identification of proper Gram stain reactions difficult.
Gram-negative organisms may not ______ properly.
Smears that are too thick or viscous may retain too much primary stain, making the identification of proper Gram stain reactions difficult.
Gram-negative organisms may not decolorize properly.
Cultures older than 16 to 18 hours will contain living and dead cells. - how does this affect gram stain?
Cells that are dead will be deteriorating and will not retain the stain properly.
What kind of stain is a gram stain?
It is used to differentiate between gram positive organisms and gram negative organisms. Hence, it is a differential stain.
LPS in gram -ve
LPS in gram -ve only – recognized by TLR4/CD14
When gram -ve cells become lysed lipid A is more freely available for recognition with immune cells
This causes a release of inflammatory cytokines: TFN-alpha, nitric oxide
What are the main differences between gram negative and gram positive organisms?
Peptidoglycan cell wall: Gram positive has thick. Gram negative has thin.
Flagellar basal body rings: Gram positive has two. Gram negative has four.
Outer antigen: Gram positive: Teichoic acid. Gram negative: Lipidpolysaccharide
Periplasmic space: Gram positive does not have. Gram negative has.
Porin channel: Gram positive does not have. Gram negative has.
Lysozyme and penicillin attack: Gram positive are sensitive. Gram negative are resistant.
What bacteria do not gram stain well?
Mnemonic: These Rascals May Microscopically Lack Color
- Treponema
- Rickettsia
- Mycobacteria
- Mycoplasma
- Legionella pneumophila
- Chlamydia
Why does the following bug not gram stain well?: Treponema
Too thin to be visualized
Why does the following bug not gram stain well?: Rickettsia
Intracellular parasite
Why does the following bug not gram stain well?: Mycobacteria
high-lipid-content cell wall
Why does the following bug not gram stain well?: Mycoplasma
No cell wall
Why does the following bug not gram stain well?: Legionella pneumophila
Primarily intracellular
Giemsa stain is a gold standard staining technique that is used for both thin and thick smears to examine blood for malaria parasites, (T/F)
True
What microbes can be stained with: Giemsa’s
- Borrelia
- Plasmodium
- Trypanosomes
- Chlamydia
Period Acid-Schiff (PAS) - purpose
Demonstrate the fungal hyphae and yeast-forms of fungi in tissue samples to identify Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans
What microbes can be stained with: Ziehl-Neelsen/ Acid fast stain
Acid-fast bacteria namely Mycobacteria (have high MW lipid cell walls that repel gram staining, and are slow-growing)
Acid fast stain for Mycobacteria - Results
Mycobacteria show up as beaded, slender, pink rods, usually stuck together in clumps
What microbes can be stained with: Ziehl-Neelsen/ Acid fast stain
Binds strongly only to bacteria that have waxy material in the cell wall is not decolorized by acid-alcohol
Acid-fast bacteria: MY LEG IS NOW CREEPY RED n BRUISED Mycobacteria (have high MW lipid cell walls that repel gram staining, and are slow-growing) Legionella Isospora Nocardia Cryptococcus Rhodococcus n Brucella
What microbes can be stained with: Silver stain
- Fungi
- Pneumocystis Pneumonia
- Legionella
- Treponema
Types of stains
- Simple (methylene blue,carbolfuschsin,crystal violet and safranin)
- Differential (Gram, Acid-fast)
- Special (Negative, Endospore and Flagella)
Stains consist of:
a Positive and Negative Ion
Chromophore
part of a molecule responsible for its colour
Basic Dye
The Chromophore is a Cation
Acid Dye
The chromophore is an anion
Negative Staining
Staining the background instead of the cell
Some basic dyes used in class are:
Methylene blue
Crystal Violet
Saffranin Red
Simple Stain
Use of a single basic dye
A mordant
May be used to hold the stain or coat the specimen to enlarge it.
Differential Stains
Used to distinguish between bacteria
- gram stain and acid-fast stain
- Require multiple stains
Gram positive bacteria tend to be killed by penicillin and detergents (T/F)
True; Gram positive bacteria tend to be killed by penicillin and detergents
Gram Negative Bacteria are less resistant to antibiotics (T/F)
False; Gram Negative Bacteria are more resistant to antibiotics
Special Stains
Used to distinguish parts of cells in microrganisms.
- Capsule Stain
- Endospore Stain
- Flagella Stain
Negative Staining for Capsules
Definition
- Used to demonstrate the presence of capsules
- Capsules don’t accept most stains = appear unstained
- Stand out against contrasting background
Endospore Staining
- Used to detect presence of endospores in bacteria
- Primary stain (machita green) with heat decolorizes cells red
- Endospores turn green when machita green with heat is used
- Endospores turn red/pink when counterstain safranin is used
Flagella Stain - define and explain process
Used to demonstrate the presence of flagella
Mordant is used until it becomes visible then is stained with carbolfuchism
List categories and types of Stains
Simple (methylene blue,carbolfuschsin,crystal violet and safranin)
Differential (Gram, Acid-fast)
Special (Negative, Endospore and Flagella)
