Week 3 - Bacteriology Flashcards
How does a gram strain work?
- Bacteria are fixated
- Crystal violet stain is added which binds to bacteria’s peptidoglycan wall
- Iodine treatment is added to stained bacteria
a) Gram positive bacteria have a very thick peptidoglycan wall so violet stain is embedded deeply = will not come off
b) Gram negative bacteria has a very thin peptidoglycan wall so stain will wash off - Counter stain such as safranin is added and will stain the empty gram negative bacteria pink whilst gram positive bacteria will remain violet
What are biochemical tests to identify bacteria from an infection? Staphylococcus aureus:
Background context:
a) Using oxygen and reactive oxygen species to classify bacteria
b) Obligate aerobes will grow at top of tube whilst obligate anaerobe will grow on bottom
c) Obligate aerobe will have SOD and catalase whilst obligate anaerobe will not have either
1. Gram stain
2. Catalase test:
a) Identifies gram positive organisms that produce the enzyme catalase
i) If catalase positive, organism is Staphylococcus
b) This is performed by giving organisms hydrogen peroxide and if the bacterial organism contains catalase it will form bubbles - this is because it is breaking down the H2O2 into H2O and O2
3. Coagulase test:
a) Identifies organisms that contain enzyme coagulase (important in clotting blood plasma)
b) Performed on catalase positive (gram +) species to distinguish coagulase positive S. aureus - coagulase is a virulence factor that forms clots around infection to protect it from phagocytosis by the immune system
c) Add bacteria to coagulase tube - if its coagulate positive it will form a clot, if negative will remain liquid
What are biochemical tests to identify gram positive bacteria?
- Motility Agar - test for flagella
- Mannitol Salt Agar
- Catalase test
- Gram staining
- Coagulase test
- Blood Agar Plates
- Nitrate Broth
- Starch hydrolysis test
What are biochemical tests to identify gram negative bacteria?
- Sugar broth with Durham tubes
- Nitrate broth
- Motility Agar
- MacConkey Agar
Describe the starch hydrolysis test:
- Used to identify bacteria that can hydrolyse starch using enzymes a-amaylase and oligo-1,6-glucosidase
a) Often used to differentiate species from the genera Clostridium and Bacillus - Carbohydrate starches such as amylose and amylopectin are too large to pass through bacterial cell wall
a) If starch is provided, bacteria must secrete enzymes outside cell to break down starch into smaller subunits to allow it to pass through bacterial cell wall - Iodine is added to agar which forms a dark brown/black colour
a) If hydrolysis of the starch occurs, it forms a clear zone around the bacteria as the starch is absorbed
Describe sugar broth with Durham tubes (Carbohydrate fermentation):
- Differential medium to test bacterial ability to use glucose (or other sugar) as an energy source
a) Commonly used to identify gram -ve enteric bacteria - Bacteria are added to sugar broth in a Durham tube also containing indicator
a) If bacteria can metabolise the sugar - phenol red indicator changed to yellow indicating pH change
3, Some bacteria produce a gas (e.g. CO2) which can also be detected in Durham tube
Describe nitrate broth:
- This is a differential medium to determine whether a bacteria can reduce nitrate to nitrite or another nitrogenous compound
a) The action of the enzyme nitratase is important in the identification of both Gram-positive and Gram-negative species - Bacteria is incubated in a tube with nitrate broth
a) After incubation, nitrate I and nitrate II are added and if nitrite is present, it will react with nitrate I and nitrate II to form a red compound = positive result
b) If no red colour forms, this indicates that either there was no conversion of nitrate to nitrite (negative result) or nitrate was reduced to nitrite and then reduced further into an undetectable form of nitrogen (positive result) - To distinguish between negative and positive result when no colour change occurs, zinc is added to broth
a) Zinc will convert any remaining nitrate to nitrite and nitrite will react with nitrate I and nitrate II to produce red pigment (negative result)
b) If Zinc produces no colour change, then nitrite was converted to another undetectable form of nitrogen (positive result)
Describe cell division in regards to bacterial genetics and antibiotic resistance:
- Cell division is a very rapid process (some bacteria can be every 20-40 minutes
a) Bacterial growth and division are important targets for antimicrobial agents - Cell division involves:
a) Segregation of replicated genomes
b) Formation of a septum (invagination of cell)
c) Division to give separate daughter cells
Describe bacterial DNA replication:
- DNA replication:
a) Triplet base sequence is universal to code for amino acids
b) Ribosomes assemble proteins
c) Each gene is the basis for a mRNA copy made by transcription, and that mRNA code is translated as a chain of amino acids - Organisation of prolaryotic DNA
Describe bacterial DNA replication:
- DNA replication:
a) Triplet base sequence is universal to code for amino acids
b) Ribosomes assemble proteins
c) Each gene is the basis for a mRNA copy made by transcription, and that mRNA code is translated as a chain of amino acids - Organisation of prokaryotic DNA
a) Single circular chromosome of double-stranded (ds)DNA - chromosome is tightly packed as a supercoil
b) Also have extrachromosomal dsDNA that is carried as a plasmid
Describe bacterial DNA replication:
- DNA replication:
a) Triplet base sequence is universal to code for amino acids
b) Ribosomes assemble proteins
c) Each gene is the basis for a mRNA copy made by transcription, and that mRNA code is translated as a chain of amino acids - Organisation of prokaryotic DNA
a) Single circular chromosome of double-stranded (ds)DNA - chromosome is tightly packed as a supercoil
b) Also have extrachromosomal dsDNA that is carried as a plasmid
i) Plasmid is not located on bacterial chromosome (separate) and replicate independently of chromosome
ii) Genes on plasmids are for plasmid replication and they also often give a phenotypic advantage to bacteria e.g. antibiotic resistance, toxin gene for virulence
iii) Plasmids are not essential for survival
What are the steps of DNA replication?
- Diagram
Describe transcription and translation:
- YouTube video
Why is gene expression in bacteria highly regulated?
- To be able to switch genes on or off to allow quick adaptation to changes in the environment
2.
Why is gene expression in bacteria highly regulated?
- To be able to switch genes on or off to allow quick adaptation to changes in the environment
a) Change in nutrients = change metabolic pathway (glucose depletion move to lactose), increase/decrease growth, dormancy
b) Change in location = ability to attach to something can be altered to attach to new location, motility increase/decrease
c) Increase virulence e.g. toxin gene activation
