Prac Flashcards
Why do we measure cell number at 600nm?
The cells would not be killed at this wavelength
What wavelength to measure protein concentration?
205nm
An isolated colony
In an isolated colony, all the cells will be genetically identical and will represent a pure culture
Colony morphology
- Form: circular, irregular, filamentous or rhizoid
- Elevation: raised, convex, flat, umbonate or crateriform
- Margin: entire, undulate, filiform, curled or lobate
Colony classification
Motile bacteria may produce larger colonies due to bacterial spread
Gram stain steps
- Fix bacteria by passing it through a bunsen flame
- Stain with crystal violet, leave for 1 minute then pour off and rinse with H20
- Cover with iodine and leave for 1 minute, then pour off and rinse with H20
- Rinse with alcohol until the colour doesn’t wash away and wash with H20
- Counterstain with safranin and rinse with H20
Lactic acid bacteria
The order Lactobacillales, which includes Lactobacillus, Leuconostoc, Pediococcus, Lactococcus and Streptococcus, in addition to Carnobacterium, Enterococcus, Oenococcus, Tetragenococcus, Vagococcus, and Weisella
They obtain energy only from the metabolism of sugars, lactic acid bacteria are restricted to environments in which sugars are present
They are used in manufacture of dairy products
S. pneumoniae, S. pyogenes and S. agalactiae; the intestinal group D streptococci S. faecalis and S. faecium; and economically important starter species S. thermophilus
Probiotics
- The most common probiotics are the Lactic Acid bacteria, but they also include other bacteria: Bifidobacterium, Bacillus and E.Coli
Lactobacillus
- Gram positive and a facultative anaerobe
- Rod shaped, non spore forming
- They are part of the Lactic acid bacteria group (convert sugars to lactic acid)
- Commensal biofilms in the vagina and gut
Oxidase test (Distinguish between oxidase negative Enterobacteriaceae and oxidase positive Pseudomadaceae)
Positive result = purple
Pseudomonas aeruginosa
Kanamycin
Aminoglycoside
Neomycin phosphotransferase II gene (NPT II/ Neo)
It encoes for resistance to aminoglycosides
B-lactamase gene (bla)
It encodes for B lactamase that are resistant to B lactam antibiotics
It catalyses the breakdown of the B lactam ring
Minimum inhibitory concentration
The lowest concentration of an antimicrobial drug that will inhibit the visible growth of a microorganism after overnight incubation
A lower MIC value indicates that less drug is required for inhibiting growht of the organism
Drugs with lower MIC scores are more effective antimicrobial agents
Bacteriostatic
A drug that prevents bacterial growth and reproduction but does not necessarily kill them
When it is removed from the environment the bacteria start growing again
How to test MIC
- The microorganisms can be tested for their ability to produce visible growth on a series of agar plates (agar dilution), in tubes with broth (broth dilution) or in microplate wells of broth (broth microdilution)
Kirby- Bauer antibiotic testing
It is a method to determine the sensitivity of microorganisms to specific antimicrobial drugs: greater drug efficacy yields larger microbe- free zones surrounding the drug-containing disks after overnight growth
Zone of inhibition
This is an area of media where bacteria are unable to grow, due to presence of a drug that impedes their growth
A larger zone of inhibition around an antibiotic containing disk indicates that the bacteria are more sensitive to the antibiotic in the disk
Antimicrobial resistance
Microorganism mediated antimicrobial resistance can be intrinsic or acquired
Intrinsic- innate
How to do MIC test
- Plat a known colony of bacteria on a plate overnight
- Add circle disks around the plate containing antibiotic
- Incubate overnight
- Measure the zone of inhibition
Biofilms, persisters and antibiotic tolerance
- Biogilms and persisters are bacterial communitites responsible for chronic diseases and antibiotic tolerance
- Biofilms are aggergates of microbial cells that form to avoid antimicrobial agents or attack by the immune system
- Persisters are non-growing microbial cells that can tolerate antibitoic treatement
- Biofilms and persisters are responsible for chronic bacterial infections and recurrent disease
Coagulase test
- It is an enzyme that clots blood plasma
- It is tested on gram positive, catalase positive species to identify the coagulase positive S.aureus
- It is a virulence factor of S. aureus
- The formation of clot around an infection cause by this bacteria can protect it from phagocytosis
Optochin sensitivity testing
Bile soluble
- It is used to distinguish between organisms sensitive to the antibiotic optochin and those not
- It is used to distingish Streptococcus pneumoniae (optochin sensitive) and other a-hemolytic streptococci (optochin resistant)
Mannitol Salt Agar
- The high concentration of salt slects for members of the genus Staphylococcus as they can tolerate high saline levels
- It contains the sugar mannitol and the pH indicator phenol red
- If an organism can ferment mannitol, an acidic byproduct is formed that will cause the phenol red in teh agar to turn yellow
- Most pathogenic staphylococci, will ferment mannitol
Bile esculin agar
- It is used to identify enterococci and group D streptococci
- It is based on the ability of an organism to hydrolyse esculin
- Bile esculin agar contains bile salts to inhibit the growth of gram positive organisms other than enterococci and group D streptococci
- It also contains nutrients: esculin and ferric citrate
- When an organism hydrolyses the glycoside esculin to form esculetin and dextrose, the esculetin reacts with ferric citrate to produce a black iron complex
- A positive result= black
Acid fast stain
- It is used to identify acid- fast organisms such as Mycobacterium
- As the cell wall is so resistant to most compounds, acid-fast organisms require a specil staining technique
- The primary stain used in acid- fast staining, carbolfushin, is lipid- soluble and contains phenol, which helps teh stain penetrate the wall
- It is aided by heat
- THe smear is then rinsed with a strong decolouriser which strips the stain from all non-acid fast cells
- The decolourised non acid fast cells take up the counterstain
- Not acid fast= purple
- Acid fast= pink
Thayer Martin agar
- It is used for culturing and isolating pathogenic Neisseria bacteria as the medium inhibits the growth of most other microorganisms
Components:
- Vancomycin which kills most gram positive organisms
- Colistin, which kills most gram negative organisms excpet Neisseria
- Nystatin, which kills most fungi
Intrinsic resistance to Vancomycin
- Lactobacillus
ONPG
- It differentiates N. lactima from N. gonorrhoeae
- Positive result = yellow (E.coli)
- Negative result (colourless)
New antimicrobials
- Target teichoic acid biosynthesis
- Produce ribosomal inhibitors to target protein synthesis
- Target outer-membrane transporters with protein epitope mimetics
