1.4 Microbiology

Question 1

What are the 3 PHYSICAL differences between bacteria that we look at? One of the physical features, depending on the structure, can impact the _____ techniques/methods used to identify the bacteria.

Answer 1

a) Size
b) Shape
c) Cell wall structure - this physical feature depending on the cell wall structure can impact staining methods used to identify the bacteria.

Question 2

Give 2 GENETIC differences that we look at between bacteria… The last point has to do with the type of proteins found on their surface.

Answer 2

a) Varying metabolic features
b) Antigenetic properties - i.e. the type of proteins found on the surface of the bacteria - those proteins are called ANTIGENS.

Question 3

A genus (a taxonomic category that’s found above ‘species’ & below ‘family’) can have 1 of 3 main SHAPES that can be used to identify/categorise the bacteria. The name of the genus can indicate that shape. What are the three types of genus names we look at and give the name of the shape.

Answer 3

a)BACILLUS - Rod shaped bacteria
b)COCCUS - Spherical shaped bacteria
c)SPIRILLIUM - Corkscrew-shaped/Spiral shaped

Question 4

Apart from the shape itself allowing us to categorise bacteria, we can also identify bacteria from the way they group/cluster with each other (bacteria of the same kind).
There are 3 types of ways bacteria spherically shaped cluster/organise themselves. Identify the names and whether they organise in chains or large clusters.

Answer 4

a) StreptoCOCCUS - They tend to group in CHAINS. The spherical shaped bacteria form chains.
b) StraphyloCOCCUS - They tend to group in CLUSTERS. The spherical shaped bacteria cluster around each other.
c) DiploCOCCUS - Bacteria are in PAIRS.

Question 5

Another method of bacteria identification is the ‘Gram Stain Reaction’. What is the ‘Gram Stain Reaction’?

Answer 5

It’s another method of bacteria identification microbiologists use to distinguish between Gram +VE bacteria & Gram -VE bacteria.

Question 6

Following on from the last card. Why do these two types of bacteria (Gram +VE and Gram -VE) require different types of staining techniques? Simple answer.

Answer 6

Because it’s dependent on the chemical composition of the bacteria’s cell wall.

Question 7

What colour do Gram -VE bacteria appear compared to Gram +VE? Why is this? Think about the types of stains used…

Answer 7

Gram +VE bacteria appear a purple/violet colour due to the stain used, ‘crystal violet’.
Gram -VE bacteria appear a red colour due to the COUNTER stain used, ‘red safranine’.

Question 8

Why can the crystal violet stain bind more effieciently to the cell wall of Gram +VE bacteria compared to Gram -VE? Think about the layer that Gram -VE bacteria have that Gram +VE don’t.

Answer 8

Because Gram +VE bacteria lack an outer lipopolysaccharide layer that’s found in Gram -VE bacteria.

Question 9

What is the significance of the outer lipopolysaccharide layer found only in Gram -VE bacteria - i.e. It does what against lysozyme enzymes & penecillin? It does what against crystal violet dye?

Answer 9

The cell wall of Gram -VE bacteria is supplemented by a lipopolysaccharide layer that PROTECTS the bacteria from lysosome enzymes and penecillin as well as excluding the crystal violet dye!

Question 10

If the Gram +VE bacteria lack a supplementary lipopolysaccaride layer what does this make Gram +VE bacteria succeptible to? 2 things that the lipopolysaccharide layer protects against in Gram -VE bacteria…

Answer 10

They’re succeptible to PENECILLIN & LYSOZYME ENZYMES.

Question 11

What is a lysozyme enzyme? Why are they membrane bound in our cells? Why are they essentially a threat to bacteria?

Answer 11

Lysosyme enzymes are DIGESTIVE enzymes and are membrane bound to protect surrounding organelles. They’re a threat to bacteria because the enzyme HYDROLYSES the bonds holding the peptidoglycan layer together.

Question 12

What does penecillin do to the cell wall’s (what does it prevent ?) of Gram +VE bacteria? Penecillin ____ the cell wall of Gram +VE bacteria. Because Penecillin _____ the cell wall it makes the bacterial cell suceptible to LYSIS/BURST due to water uptake…

Answer 12

Penecillin PREVENTS the bonds inter-linking the peptidoglycan molecules. Penecillin WEAKENS the cell wall of Gram +VE bacteria.

Because the cell wall is weakened by penecillin the uptake of water can BURST the cell wall - cell can’t hold itself together due to weakening of cell wall…

Question 13

If the lipopolysaccharide layer in Gram -VE bacteria protects the bacteria from PENECILLIN how do we protect against Gram -VE bacteria?

Answer 13

We use another type of antibiotic that impacts the bacteria’s ability to synthesise proteins.

Question 14

If we use the antibiotic that impact’s the Gram -VE bacteria’s ability to synthesise proteins won’t that impact the ukaryotic cell’s ability to also synthesise proteins?

Answer 14

NO, because the machinery responsible for protein synthesis in eukaryotes is different from bacteria!

Question 15

a) What is the name of the couter stain used for Gram -VE bacteria?
b) What is the name of the stain used initially that will lead to a purple colour to appear on Gram +VE bacteria?

Answer 15

a) Red Safranine
b) Crystal Violet

Question 16

What do we use to wash off the stain?

Answer 16

Alcohol - it’s a decoulouriser. Removes the colour from Gram -VE bacteria making them colourless. Can’t do this to Gram +VE bacteria though - purple remains…

Question 17

What do we use to help the crystal violet stain bind to the peptidoglycan strongly?

Answer 17

IODINE - it’s a MORDANT.

Question 18

MOVING ON FROM CLASSIFICATIO&raquo_space;>

Question 19

Bacteria undergo what to reproduce? NOT ‘mitosis’… Define that term…

Answer 19

Bacteria UNDERGO ‘Binary fission’.
Binary fission is the replication of the circular DNA and plasmids and is the division of the cytoplasm forming two IDENTICAL daughter cells.

Question 20

What 5 things do bacteria/microorganisms require for growth? The next few cards will look at testing you on each…

Answer 20

a) Nutrients
b) Growth Factors (i.e. vitamins and mineral salts such as Na+)
c) Temperature
d) pH
e) Oxygen

Question 21

Bacteria require NUTRIENTS that can be supplied in a ______.

Answer 21

Nutrient Medium

Question 22

This nutirent medium/media can be a liquid called a _____ or in solid form called _____.

Answer 22

a) Liquid from called ‘Nutrient BROTH’
b) Solid form called ‘AGAR’

Question 23

What do nutrient mediums/media provide bacteria? 3 THINGS…

Answer 23

a) Carbon & an energy source - gluecose
b) Nitrogen to allow the bacteria to synthesise amino acids
c) Water

Question 24

The SECOND requirement for culturing microorganisms is providing growth factors such as? 2 THINGS…

Answer 24

a) Vitamins
b) Mineral salts - Na+ & PO3 4- etc…

Question 25

The THIRD requirement is a suitable temperature. Why is this key to culturing microorganisms?

Answer 25

Bacterial chemical reactions (i.e. their metabolism) are controlled/facilitated by enzymes. Thus, these enzymes require certain operating temperatures to ensure they're working fast/efficiently but also the temp. must NOT be over the OPTIMUM level or the enzymes are prone to DENATURINg/DENATURATION.

Question 26

The FIFTH requirement is OXYGEN (we've skipped pH). a) What are bacteria who NEED oxygen called? b) What are bacteria who CAN survive without oxygen called? c) What are bacteria who CANNOT survive in the presence of oxygen called?

Answer 26

a) Obligate aerobes b) Facultative aeorbes c) Obligate anaerobes

Question 27

There are 5 reasons why many bacteria CANNOT be cultured in the conditions we just described/listed. Name and explain them...

Answer 27

a) Some bacteria are mutualistic (i.e they require the presence of another species in order to thrive). b) Some are INTRACELLULAR parasites c) Simply have very SPECIFIC growth requirements d) Very long generation times e) Poisoned by media components.

Question 28

There are 4 ways to DESCRIBE media:

Answer 28

a) Defined - i.e. we know what the ingredients present in the growth medium are... b) Undefined - i.e. we don't know what some of the media's components are because they include yeast extract or beef peptone. c) A selectve medium - i.e. a medium that allows only certain bacteria to grow. E.g. a selective media that only allows Gram -VE bacteria to grow... d) A COMPLETE MEDIUM - All the chemicals required to support growth are present...

Question 29

PRINCIPLES OF ASEPTIC TECHNIQUES >>>

Question 30

Bacteria & fungi are put on a nutrient media/medium which is designed to supply them with the nutritional and physical requirements they require for growth. BUT, to do this it must all be done under ASEPTIC CONDITIONS. Define 'ASEPTIC CONDITIONS'.

Answer 30

These are laboratory requirements that maintain sterility of the apparatus being used as well as the immediate environment where the culture preperation is taking place. It PREVENTS contamination.

Question 31

What do we do to the apparatus before & after usage?

Answer 31

We sterilise the apparatus to PREVENT INITIAL contamination.

Question 32

What do we do to the culture vessel's neck before using it?

Answer 32

a) We flame the neck of the culture vessel before and after using it.

Question 33

What do we do to the environment where we are operating/working on growing the culture?

Answer 33

We sterilise the environment using a disinfectant.

Question 34

Describe the 6 step innoculation process. Include all details.

Answer 34

1. Hold the culture bottle/vessel in one hand; open the cap with your little finger. Don't place the cap down. 2. Flame the mouth/neck of the bottle/vessel. 3. Pass the innoculaitng LOOP thorugh the flame until red hot. Allow to cool soon after in the AIR - i.e. don't place it down because that will lead to contamination... 4. Lift the lid of the petri dish enough so that the wire can go though. 5. Secure petri dish lid with tape. 6. Incubate at 25 degree.

Question 35

Why do we not seal (put tape) the petri dish fully?

Answer 35

Because this will create anerobic conditions that can promote the growth of pathogenic bacteria.

Question 36

METHODS OF MEASURING GROWTH >>>

Question 37

There are 2 ways the size of a liquid culture can be measured. Directly couting cells & indirectly counting cells (i.e. measuring what's called _____. For direct couting of cells what are the names of the 2 types of counts (think about live and dead cells).

Answer 37

Measuring what's called TURBIDITY using a HAEMOCYTOMETER - a cell counting chamber used origionally to quantify RBC. Viable counts - i.e. counting the number of colonies (live cells) Total counts - i.e. using a HAEMOCYTOMETER - a cell counting chamber used origionally to quantify RBC. Total counts highlight the no. of dead AND live cells.

Question 38

We spoke about measuing TURBIDITY (i.e. cloudiness) as an indirect method. When is this technique used mostly? Explain what is done to the sample of bacteria to quantify the no. of bacteria in the sample.

Answer 38

Fieldwork. To quantify the number of bacteria cells in a given sample we measure the light absorbed by a particular sample, e.g. a sample of river water. The more cloudy the water the less light shall be absorbed indicating a high bacterial count.

Question 39

A device called a _____ can be used to determine light absorbtion for a given sample. Does this method give you a viable or a total count - explain your answer.

Answer 39

COLORIMETER. A colourimeter provides a TOTAL count for a given sample becuase obviously a colourimeter cannot distinguish between viable (i.e. live cells) and dead cells in the given sample.

Question 40

Measuirng growth directly is only possible by first carrying out serial ______. This method relies on each bacterial cell forming what to provide a VIABLE count?

Answer 40

Serial DILUTIONS This method relies on the bacterial cells forming colonies with each other. A colony is a cluster of cells.

Question 41

Give the formula that allows you to find the 'dilution factor'.

Answer 41

diliution factor = 1 / conc.

Question 42

After the serial dilutions are complete 1cm^3 of each diluted sample is then _____? After that it is incubated at what temp. and for how long?

Answer 42

Spread over a large AGAR PLATE then incubated at 25 degrees for 2 days.

Question 43

What happens if the dilution is too great? What happens if the dilution is insufficient?

Answer 43

a) There shall be too FEW colonies that appear on each plate for the count to be statistically sound! b) There shall be overlapping between colonies (i.e. they merge). Leads to an accurate/underestimation of numbers.