3. Microbial techniques methods

Question 1

Describe aseptic technique

Answer 1

How to make a pure culture of specific microorganism without contamination from all the bacteria around us? Successful cultivation and maintenance of pure cultures of microorganism can be done only if aseptic technique is practiced to prevent contamination by other microorganisms. Important to cover the plate with the lid when streaking the liquid out on the plate.

Question 2

How can something be sterilized?

Answer 2

Liquids (e.g. culture media) are autoclaved our filter-sterilized to inactivate/remove microorganism. In an autoclave liquids are heated by steam (e.g. 121 degrees 15-20 min) Steam kills microorganism more efficiently than air. Liquid may also be filtred through small-pore filters (0.2-0.5 mikroM) retaining bacteria and viruses.

Question 3

What is a pure-, stock- and mixed culture

Answer 3

ONE organism = pure culture

if STORED = stock culture

> 1 typed = mixed culture

Question 4

Describe defined media.

Answer 4

Defined (chemically defined medium); sometimes minimal medium

Question 5

Describe syntetic media.

Answer 5

same as defined

Defined (chemically defined medium); sometimes minimal medium

Question 6

Describe undefined media.

Answer 6

complex medium

Question 7

Describe general media.

Answer 7

supports growth of as many organisms as possible

Question 8

Describe selective-, enrichment media.

Answer 8

Selective, enriching, enrichment medium – media used for the isolation of particular species/types. Support growth of selected organism, inhibits others.

Enriched – media that have been supplemented with highly nutritious materials such as blood, serum or yeast extract for the purpose of cultivating fastidious organism

Question 9

Describe natural media.

Answer 9

Natural – e.g. molasses, whey, fruit juice

Question 10

Describe how blood agar can indicate different hemolyses.

Answer 10

Different types of hemolyses shows different results.

alfa – the agar under the colony is dark and greenish. Streptococcus pneumoniae and some oral streptococci display alpha

beta – complete hemolysis of red cells in the medium around/under the colonies: light yellow and transparent. Streptolysin, an exotoxin, is the enzyme produced.

Question 11

Describe differential media.

Answer 11

Widley used for differentiating closely related organism or group of organism. Certain dyes or chemicals in the media, specific organism will produce characteristic changes or growth patterns used for identification or differentiation.

Question 12

Describe enrichment

Answer 12

Enrichment – highly important technique
Generally necessary for detection of food pathogens
The basic principle is selection. Often it is desired to isolate or detect bacteria that are present in very low numbers. This create a problem.
Enrichment culture solves this problem. The essence of this technique is to provide growth conditions that are very favorable for the organism of interest, and as unfavorable as possible for competing organism. May be: physical conditions, nutrient content

Question 13

Describe pre-enrichment.

Answer 13

Generally, to detect very low levels of *Enterobacteriaceae in food, the sample preparation must include pre-enrichment. Damaged or stressed cells (e.g. by heat or cold) are recovered meaning they can start to grow. Direct enrichment often too harsh for damaged cells; e.g. too low pH
A (universal) pre-enrichment broth is used. Provided enough time, sub-lethally injured cells will resuscitate – normally rich broth with good buffering capacity.
Next step is normally enrichment.

Question 14

Describe batch, fed-batch, and continuous culture

Answer 14

Batch reactor or batch cultivation
Fed only once (no more feeding after that) – the bioreactor is operating on batch mode. The bioreactor will run till completion. At the end the fermentation is terminated before a new fermentation is initiated again.

Fed batch is the intermediary model of operation, characterized by predetermined or controlled addition of nutrients into the bioreactor at certain times of fermenter operations.
Allows temporal variations in the supply of nutrients.
gives a degree of control on the process and operations of the fed batch bioreactor; can control the rate of growth of the microorganism or concertation of the biomass by controlling parameters such as frequency and concentrations of feeds. Used in industry to maintain high concentration of microorganisms reduce feedback inhibition, especially catabolite repression. Fed batch is the most popular mode in fermentation industries.

The bioreactor is fed continuously – a continuous bioreactor. A fixed volume; the amount of feed introduced into the bioreactor is equivalent to the spent volume removed from the bioreactor. Despite the ideal characteristics of the continuous bioreactor, the process itself is sensitive, and subjected to influence from various factors such as risk of contaminations, cell or biomass washouts.

Question 15

Describe different colony morphology

Answer 15

Form – what is the basic shape of the colony? E.g. circular, filamentous, etc.

Size – the diameter of the colony

Elevation – this describes the side view of a colony. Turn the petri dish on end.

Margin/border – the edge of a colony. What is the magnified shape of the edge of the colony?

Surface – how does the surface of the colony appear? For example, smooth, glistening, rough, wrinkled or dull.

Opacity – for example, transparent (clear), opaque, translucent (like looking through frosted glass), etc.

Colour – (pigmentation) – for example

Question 16

Describe a simple stain method.

Answer 16

Simple stain
- routinely used for determining numbers, size, shape and arrangement of cells.

• The dry mount is colored with color for 1-2 min before being rinsed of and observed in microscope.
• all organism are stained with the same colour
• acidic or basic dyes (methylene blue or crystal violet)

Question 17

Describe negative staining.

Answer 17

Negative staining (indirect staining with acidic dye)
Does not stain the bacteria due to ionic repulsion, but the background
bacteria will appear colorless against a dark background. No heat fixation or strongly toxic chemical used  bacteria less distorted
E.g. nigrosine

Question 18

Describe gram staining.

Answer 18

Used to se the difference between gram positiv and gram negativ bacteria.

Gram stain (differentiates 2 classes of bacteria (gram positive/gram negative))
Principle: different cell wall structure results in different staining.
Gram + stain blue due to crystal violet
Gram – stain red due to safranin

Procedure:
1) A heat-fixed smear is flooded with the basic purpule dye, usally crystal violet. Because the purple stain colors all cells, referred to as a primary stain.

2) After 1 minute, the crystal violet is drained off and washed whit distilled water. The smear is then covered with gram’s iodine, a mordant or helper. When the iodine is washed off, both gram-positive and gram-negative bacteria appear dark violet or purple.
3) Next the slide is washed with alcohol (95% ethanol) or an alcohol-acetone solution. This solution is a decolorizing agent which removes the purple from the cells of some species but not from others. When the procedure is carried out, the slide is hela at an angle and 95% ethanol is poured until the draining solution no longer has a purple tint.
4) The alcohol is now rinsed off with distilled water and the slide is then stained with safranin, a basic red dye known as the counter stain. The staining process is allowed to be carried out for 2-3 minutes. The smear is washed again, heat dried and examin microscopically.

Explanation of gram-staining
G+ bacteria have a thicker peptidoglycan (disaccharides and amino acids) cell wall than gram negative G- bacteria. G- bacteria contain a layer of lipopolysaccharide (lipids and polysaccharides) as part of their cell wall. When applied to both G+ and G- cells, crystal violet and then iodine readily enter the cells. Inside the cells, the crystal violet and iodine combined to form the crystal violet-iodine (CV-I) complex. The CV-I complex is larger than the crystal violet molecules and cannot be washed out of the intact peptidoglycan layer of gram-positive cells by alcohol. G+ cells retain the colour of the crystal violet dye. In G- cells, however the alchol wash disrupt the LPS layer -> CV-I complex is washed out through the thin layer of peptidoglycan.  G- cells are colorless until conterstained with safranin, after which they become pink.

Question 19

What is CFU?

Answer 19

colony forming units.

Diluted cells is spread on plate, number of colonies are counted to get the CFU.

Question 20

Describe Turbidity (OD) measurement.

Answer 20

Turbidity – The amount of scattered and absorbed light is proportional to amount of cells.

dilatated cell solutions: not good to have to densed solution

Lower wavelength sensitive but when using complex media cause you get a interference

Better: log scale on Y-axes -> when you get a straight line(along the x axis) you know that the bacteria is growing at theire max rate.
generating a standard curve

Question 21

Describe thymidine incorporation.

Answer 21

3H-thymidine incorporation into DNA is widely used to e.g. estimate rates of bacterial growth and secondary production in aquatic system.

The principle:
[3H]-thymidine is a labelled DNA precursor (will not be used in RNA)
When cells divide, DNA is replicated and precursors incorporated
[3H]-thymidine is added to a culture or other sample, proliferating cells will take up and incorporate [3H]-Thymidine
The amoint of [3H]-Thymidine incorporated into the DNA is measured with scintillation counter.
The level of the radioactive signal depends on the proliferation rate – the higher the proliferation rate, the more radioactive DNA will be synthesized.
Dna synthesized is more or less constant? While Protein syntatist is regulated depending on the conditions.

Question 22

Describe ATP bioluminescence instrument.

Answer 22

Well-established technology
Instant indication of the hygiene status of product contact surfaces
More problematic to use directly on food because of reaction with non-microbial ATP.

Reaction:
Specific reaction of ATP (in living cells) and enzyme complex (luciferin/luciferase)
Release fluroscent ligh depending on the amount of ATP
Number of cells can be calculated based on amount of ATP
Rapid (1-2 min)
Used in dairy product for estimating total bacteria count

Question 23

Describe immunomagnetic separation IMS

Answer 23

Challenge: to separate the organism of interest from a sample high in background (examples some of the bacteria may not be pathogenetic
IMS – developed to accomplish this rapidly and reliably
Can be used to quantify pathogenicity of food, blood or feces.

Principle: Specific antibodies against the desired target are linked to the beads
beads precoated with secondary antibody that has specificity for the primary antibody
2 step detection because of the use of 2 different antibodies.
Coated beads added to a sample suspension in a tube will bind to the target.
The beads-target complexes can be separated from the environment to the side of the tube using a strong magnet and recovered or removed depending on the object of the method.

Question 24

Describe serotyping.

Answer 24

Serotyping (serological typing) – strains from the same species can differ in the antigens expressed on the cell surface. Reacts with different antibodies.
The salmonella genus for example, has been determined to have over 4400 serotypes
Serotyping is applied to numerous species that express different serotypes, such as: Escherichia coli, pseudomonas aeruginosa, salmonella species, shigella species, Yersinia and vibrio cholerae

Question 25

Describe ELIZA.

Answer 25

Sandwich ELISA – capture antibody immobilized onto solid surface of e.g. microtiter plate
Blocking with e.g. bovine serum albumin to diminish non specific binding
Sampled added, antigen binds to the antibody, the rest is removed
Second, enzyme-labelled antibody added, wash
Add fluorescent/coloured substrate

Question 26

Describe DNA hybridization.

Answer 26

Target DNA (bacterial) is attached to membrane filter (nitrocellulose)
DNA probe (known oligonucleotides) bin to target DNA
Detect by radioisotope or colorimetric techniques
Commercially available – gene trak (salmonella typhimurium, listeria monocytogenes, esherichia coli)

Question 27

Describe PCR based assay primers.

Answer 27

Primers for specific genes allow specific, rapid and sensitive detection of pathogens and toxins(you look for the gene that results in the toxins, not direct measurement.

PCR. Primers som ligger i änden på sitt mål DNA blandas med DNAt. Genom att höja temperaturen(över 94° C) kommer den dubbelsträngade DNA molekylerna brytas till esDNA. Temperatursänkning till 50-60° C kommer resultera i att primerna binder in till de esDNA, genom att höja temperaturen igen (68-72° C) kommer primerna förlänga längst DNA stranden. Genom att upprepa detta kommer man få en exponentiell tillökning av DNA fragmentet som är av intresse. Detta kommer man få först efter cykel 3, dvs där primers ändar möts. Primers ska vara 18-24 mers långa, ha ett 40-60% innehåll av G/C, 2/3 sista nukleotiderna ska vara G/C och sista ska vara G/C, primerna får inte ha en sekundär struktur

Question 28

Describe colony hybridization.

Answer 28

The idea is that the probe only bind to the complementary DNA.
Typically very sensitive. To be sure that we have DNA we could stain, different probes?

Kolonihybridisering. Man flyttar över en bit av varje koloni till ett membran eller filter där man sedan gör lysis på bakterierna och får ut DNA. Tillsätter sedan markerad DNA probe. Proben kommer binda in till den kolonin med matchande sekvens, plockar upp DNA kolonin och sekvenserar sedan den.

Question 29

Describe Quantitative-PCR /Real Time -PCR.

Answer 29

PCR products detected in real time, cycle at which fluorescence detected (Ct) inversely proportional to amount of specific DNA in sample. Multiplexering possible – different flurophores

Real time PCR. Genom att mäta signal från det dsDNA efter varje cykel kan man se hur snabbt DNAt ökar. Man letar efter CT värdet vilket är den cykel då mängden DNA överstiger ett givet tröskelvärde. Om det finns mycket DNA kommer CT nås snabbt medans om det finns lite DNA kommer det ta tid att nå värdet. Sätt att detektera virus.