lecture 4

Question 1

how do we find the microorganism that caused a disease

Answer 1

1) find the bacteria via tests (e.g. mouth swaps, blood tests, urine samples)
2) grow the bacteria in vitro
3) identify the bacteria (via a large array of diagnostic methods)

Question 2

what should you do to isolate and grow the bacteria in vitro

Answer 2

a single bacterial cell multiplies into a visible colony making it possible to separate specific pathogens from a mixed sample

Question 3

examples of diagnostic methods used to identify the bacteria

Answer 3

based on:
1) structural features
2) biochemical properties
3) antibody binding
4) infection by phage
5) DNA/RNA

Question 4

how do structural features help to distinguish bacteria

Answer 4

cell morphology, gram staining

Question 5

how do biochemical properties help to distinguish bacteria

Answer 5

• O2 dependence
• chemical resistance
• antibiotic resistance
• particular metabolic enzymes

Question 6

how does antibody binding help to distinguish bacteria

Answer 6

• serological tests (to determine if your immune system has responded to a specific infection)
• immunological assays (measures the presence or concentration of a specific molecule in blood/urine)

Question 7

how does infection by phage help to distinguish bacteria

Answer 7

observing the bacteria’s susceptibility to specific bacteriophages

Question 8

describe how gram-staining helps to distinguish bacteria

Answer 8

1. crystal violet floods the slides and stains all bacterial cells (all cells are now purple)
2. iodine reacts with crystal violet to form a crystal violet–iodine complex (CV-I) inside the cells.
3. the complex is hard to wash out and gets trapped especially well in thick peptidoglycan layers
4. alcohol/acetone is quickly added
5. gram positive - dehydrates the thick peptidoglycan layer, CV-I complex gets trapped. they stay purple
6. gram negative - alcohol dissolves the outer membrane. thin peptidoglycan cannot retain the dye complex. CV-I complex leaks out. they turn colourless
7. safranin is added: gram-negative bacteria absorb the dye → pink. gram-positive bacteria already have strong crystal violet → stay purple

Question 9

what happens to candida albicans with gram staining

Answer 9

• appear purple
• however they are NOT gram positive, as they have no peptidoglycan wall and are not a bacteria

Question 10

how do you identify candida albicans through staining

Answer 10

they grow pseudohypae which other yeasts don’t

Question 11

what is mycobacteria

Answer 11

bacteria that don’t appear gram positive or negative (colourless) due to a hydrophobic waxy coat, that repels hydrophilic gram stain

Question 12

what staining does mycobacteria undergo

Answer 12

ziel-neelsen staining

Question 13

describe how ziel-neelsen staining works

Answer 13

1) use carbolfuchsin and heat to melt the waxy layer
2) carbolfuchsin turns the mycobacteria red
3) then ethanol is used to destain
4) methylene blue is used to counter stain, which turns the other cells blue
5) we now see the red bacteria against a blue background

Question 14

how do you stain molds

Answer 14

lactophenol cotton blue stain

Question 15

describe how lactophenol cotton blue staining works

Answer 15

1) the lactic acid preserves the fungi and makes the tissue more clear
2) phenol kills the fungus, so you’re looking at dead, stable structures
3) cotton blue stains chitin in the cell walls of fungi

Question 16

describe what you would see on penicillum

Answer 16

• hyphae branches
• the branches give rise to fruiting bodies
• the fruiting bodies give rise to spores

Question 17

what is the catalase test for

Answer 17

• if bacteria is gram positive, it is most likely staphylococci or streptococci
• catalase test is used to distinguish between the two

Question 18

how does catalase test work

Answer 18

• only staphylococcus has catalase
  1) you put the bacteria on the slide then add hydrogen peroxide to it
  2) hydrogen peroxide is the substrate for the catalase
  3) catalase converts the peroxide into water and oxygen, producing bubbles
  4) streptococci don’t have catalase, so they won’t form bubbles

Question 19

what is the coagulase test for

Answer 19

differentiating between staphylococcus aureus or other staphylococcus

Question 20

how does coagulase test work

Answer 20

• only staphylococcus aureus has coagulase
  1) coagulase clots blood
  2) if serum is added and a clot is formed, then the bacteria is staphylococcus aureus
  3) other staphylococci are grouped as CONS - coagulase negative staphylococci

Question 21

what is the oxidase test for

Answer 21

to find out if bacteria is aerobic or anaerobic

Question 22

how does oxidase test work

Answer 22

• presence of cytochrome c oxidase shows that its an obligate aerobe bacteria
  1) a 4x4 slide contains an oxidase agent in it
  2) bacteria is added to the slide
  3) if the enzyme is present in the bacteria, it will turn the slide blue

Question 23

examples of oxidase positive bacteria

Answer 23

• pseudomonas
• neisseria
• moraxella
• campylobacter

Question 24

how does microbact strips test work

Answer 24

• a strip with lots of wells, each containing different substrates or enzymes
• the bacteria is incubated, then you look for how your bacteria reacts with each well
• e.g. glucose will be in one of the wells
• bromothymol blue is a pH indicator which will show if acid was produced from glucose fermentation
• if the bacteria can ferment glucose, it produces acid which lowers the pH of the well and the BTB turns yellow
• if not, the BTB remains blue

Question 25

how does the index work in a microbact strip

Answer 25

1) divide the 12 wells into 4 groups of 3 2) in each group, assign the weights (1, 2, 4) to the wells 3) add the numbers only for positive wells; negative wells = 0 4) combine the 4 digits from the 4 groups to give a 4-digit index 5) look up that 4-digit code in the Microbact table → identifies the bacterium

Question 26

what is selective agar

Answer 26

agar that contains chemicals that allow only certain types of bacteria to grow while inhibiting others

Question 27

what is differential agar

Answer 27

- contains indicators or substrates that make different bacteria look different based on their metabolism. - all bacteria might grow, but their appearance changes depending on what they do

Question 28

sabouraud agar properties

Answer 28

- selective for fungi - non-differential - low pH suppresses growth of most bacteria

Question 29

eosin-methylen blue agar (EMB) properties

Answer 29

- selective for gram negative bacteria as its aniline dyes are toxic to gram positive bacteria - differentiates lactose fermenters: - pink = weak/moderate, green = strong/rapid

Question 30

MacConkey agar properties

Answer 30

- selective for intestinal pathogens as its bile salts inhibit non-enteric (non-intestinal) bacteria - differentiates lactose fermenters: - pink = fermentation, colourless = no fermentation

Question 31

blood agar properties

Answer 31

- growth of fastidious bacteria (microbes that are difficult to grow in the laboratory) - differentiates for hemolytic reactions (bacteria that carry hemolysis vs those that don't)

Question 32

mannitol salt agar properties

Answer 32

- selective for haloduric bacteria (staphylococci) - differentiates mannitol fermenters into yellow - s. aureus is the only staph that ferments mannitol

Question 33

bile-esculin agar properties

Answer 33

- selective for enteric bacteria: oxgall inhibits non-enteric bacteria - differentiates for bacteria that can hydrolyse esculin: hydrolysis gives a dark brown colour

Question 34

what is the antibiotic susceptibility test

Answer 34

- you place antibiotic discs around a bacterial lawn then incubate it - if bacteria is resistant to the antibiotic, it won't form an empty ring around the disc and still grow around it - if bacteria isn't resistant, it will form an empty ring around the disc

Question 35

what is cell agglutination

Answer 35

1) you add antibodies to cells that specifically recognise antigens on the cell surface 2) if the antigen is present, the antibodies bind to multiple cells at once, linking them together 3) this creates a visible clump

Question 36

what is latex bead agglutination

Answer 36

1) coat latex beads with either antibody or antigen 2) mix the beads with the sample 3) if the target is present, beads link together to form visible clumps 4) if target is absent, beads stay dispersed, solution looks smooth

Question 37

what is a western blot

Answer 37

- a laboratory technique used to detect specific proteins in a sample 1) proteins are taken from cells, tissues, or bacteria 2) proteins are loaded onto a gel, and gel electrophoresis separates the proteins by size 3) smaller proteins move faster, larger ones slower 4) you blot the proteins onto a filter to immobilise them 5) then you add specific antibodies to bind to your protein of interest 6) you add a secondary antibody that binds to the primary antibody and is linked to an enzyme or dye 7) when a substrate is added, the enzyme produces a visible cue (e.g. colour change)

Question 38

what is ELISA

Answer 38

1) microplate wells are coated with antigen or antibody 2) the sample is added to the well. if the target is present, it binds to the coating 3) a secondary antibody with an enzyme is added. it binds to the target bound in the well 4) unbound antibodies are washed away, leaving only the specific complex 5) the enzyme reacts with a chemical substrate → produces a colour change 6) more binding → stronger color

Question 39

what is immunofluorescence microscopy

Answer 39

- lets you see specific molecules inside cells by using antibodies linked to fluorescent dyes - you can look under a microscope to see the fluorescence

Question 40

what is RFLP

Answer 40

- molecular biology technique used to detect differences in DNA sequences between individuals or organisms - not used much anymore

Question 41

how does RFLP work

Answer 41

1) DNA is taken from the cells you want to study 2) restriction enzymes cut DNA at specific sequences 3) if there's a mutation in the DNA at a cutting site, the enzyme may cut differently, producing fragments of different lengths 4) fragments are separated by gel electrophoresis 5) the different sizes can mean infection, mutation, strain difference etc

Question 42

how does PCR work

Answer 42

1) DNA is heated and denatured, seperating the double-strands into single strands 2) the mixture is cooled 3) short DNA primers (designed to match the target sequence) bind to the single-stranded DNA 4) the temeprature is raised for Taq polymerase 5) Taq polymerase synthesises new DNA, starting from the primers 6) this is repeated, resulting in millions to billions of copies of the target DNA

Question 43

how does in situ hybridisation work

Answer 43

1) a short DNA or RNA sequence complementary to the target sequence is used 2) the DNA probe is labeled with a fluorescent dye or an enzyme that produces colour when substrate is added 3) the probe binds to its target inside the cell 4) wash to remove unbound probes 5) detect the fluorescent probe via a fluorescence microscope, or the enzyme-linked probe via a normal light microscope