Enumerating Microbes

Question 1

what can we not measure without knowing the amount of microbes present ?

Answer 1

• small increases in cell number (cell growth)
• effectiveness of control procedures (cell death)
• contamination levels (microbial load)

Question 2

what are some of the different methods used to determine how many microbes are present ?

Answer 2

• counting the physical number of cells (microscopy): does not discriminate between live or dead cells
• directly measuring the cell mass (dry weight): does not discriminate between live or dead cells
• measuring some components of the cell (biochemical assay): assumes an average value per cell
• measuring the turbidity of a culture (optical density ): useful but inaccurate estimate
• counting the number of cells that can grow into a colony (viable count): discriminates between live or dead cells and is very sensitive but requires full understanding of cell physiology

Question 3

what is a haemocytometer ?

Answer 3

it is a specialised microscope slide that has grids etched onto the surface of the sample holder

Question 4

when counting directly from a microscope what magnification does the bacterial cell need to be imaged at ?

Answer 4

x1000

Question 5

to get a statistically accurate count, how many cells does the sample need to contain ?

Answer 5

at leats 10^5 cells evenly distributed in the sample holder

Question 6

how is cell mass determined using wet weight ?

Answer 6

• all excess moisture must be removed to get a reproducible reading e.g. using filter paper wick

Question 7

how is cell mass determined using dry weight ?

Answer 7

the samples are heated to remove water
- dry weight is commonly used to estimate the amount of protein in a sample (generally 10x lower than wet weight for equivalent samples

Question 8

Does ATP measurement distinguish between live and dead cells ?

Answer 8

yes

Question 9

what is turbidity ?

Answer 9

is the cloudiness or haziness of a fluid caused by suspended solids that are usually invisible to the naked eye

• refers to the scattering of light by particles

Question 10

what 3 things can happen if light shines through a liquid culture ?

Answer 10

1. it can be transmitted through the liquid, to be detected by the sensor of a spectrophotometer
2. it can be absorbed by chemicals in the liquid
3. It can be scattered by particles in the liquid

Question 11

what is turbidity estimated from ?

Answer 11

the optical density of a culture

Question 12

what is the unit used for turbidity ?

Answer 12

optical density (OD) at a specific wavelength

Question 13

what is the optical density (or absorbance) defined as ?

Answer 13

log10(I0/IT)

• when there are no cells present l0 = lT; therefore abs = log10 (lO/lT) = log10(1) = 0

Question 14

what are some of the limitations and potential inaccuracies of using turbidity as a measure of cells ?

Answer 14

1. turbidity caused by non-biological particles can not be easily distinguished from that caused by cells
2. spectrophotometers are inaccurate at A values greater than 1 - cultures must be diluted to measure turbidity
3. cell components that absorb light affect results
4. turbidity does not distinguish between live and dead cells
5. turbidity is inaccurate for cultures with low densities
6. turbidity has a limit of detection based on the sensitivity of the spectrophotometer

Question 15

what are the 4 stages of growth phase ?

Answer 15

1. lag phase
2. exponential phase
3. stationary phase
4. death phase

Question 16

how do you measure turbidity ?

Answer 16

1. ‘blank’ the spectrophotometer with sterile culture medium
2. use wavelengths that are not readily absorbed by most cell components to measure the optical density - generally between 540 and 650 nm
3. check that nothing is produced by cells that absorbs wavelength used by comparing with a calibration curve

Question 17

how is the turbidity of a culture compared ?

Answer 17

its compared by eye with standardised tubes with pre-determined light scattering properties = MacFarland standards

• only works for cell densities above 10^7 cfu ml-1

Question 18

what is viable count ?

Answer 18

its when cells are plated on a media and left to grow into colonies and the number of colonies are counted = number of cfu in sample

Question 19

what are the advantages of viable count ?

Answer 19

the method can be very sensitive (single cells detected)

Question 20

what are the disadvantages of viable count ?

Answer 20

• colony-forming units may underestimate cell numbers because of the presence of clumps or chains of cells
• some organisms have low plaiting efficiencies; not all cells can grow into colonies
• counts usually require overnight incubation; results are retrospective

Question 21

how can you achieve countable numbers on a plate ?

Answer 21

the samples need to be diluted before plating out

Question 22

what are some of the features we need to control in the diluent ?

Answer 22

• it needs to be osmotically balanced for the type of cells

- must be temperature controlled

Question 23

what are the common used diluents ?

Answer 23

MRD = maximum recovery diluent 
PBS = phosphate buffered saline