MRA Lab Report

Microbial Risk Assessment Project

Lab report

Francis Higgins, Cliodhna McMahon and Karen Duffy

Department of Microbiology

National University of Ireland, Galway

April 2015

Experimental Question 1:

How effectively can bacteria be recovered from spiked strawberries?

Why?

From conducting a microbial risk assessment on the consumption of raw fruit and

vegetables, we found that there are many cases of Salmonellosis caused by the

consumption of the fresh produce. This is due to contamination and, without adequate

washing or preparation, this can cause illness. However, there are some unknowns.

One particularly interesting unknown was the likelihood or the rate of the produce

becoming contaminated in retail outlets or in the consumer homes, and the survival of

this contamination prior to consumption.

How?

We conducted an experiment to try and recover E. coli from fresh strawberries. As

Salmonella are highly pathogenic it was decided that E. coli would be used instead.

E.coli and Salmonella have similar growth requirements and incubation periods. We

spiked the strawberries with a known concentration of E. coli cells and the aim was to

recover these cells by conducting a wash on the strawberries and serially diluting the

wash.

List of equipment: for strawberry experiment

Fresh strawberries of equal weight/surface area

Sterile PBS diluent

Pipette pump

Petri dishes

1ml pipettes

Analytical balance

Fresh overnight culture of E. coli in nutrient broth

Experimental Design:

1 L of nutrient agar was made

1 L of MacConkey agar was made, 1 L of 1xPBS made.

Strawberries were weighed to find three strawberries of similar weight (within

.1g of each other). The three strawberries selected weighed 16.0g, 16.1g and

16.0g and were placed on individual petri dishes.

1ml of a 106 overnight culture was added to the strawberries using a pipette.

The strawberries were left for approx. 20/30 minutes.

The strawberries were then placed into Schott bottles with 90mls PBS to be

used as neats for serial dilutions.

A standard shake was performed, 25 times at a 30° arc.

Our three neats were each diluted to 10-4 by adding 1ml measurements into 9

ml aliquots of PBS. 0.1ml of each dilution was plated onto MacConkey plates.

The following controls were used: Non- spiked strawberry. PBS control (on

MacConkey), Fallout plate, MacConkey Media control

Plates were incubated at 37°C for 24 hours.

Controls for the Strawberry experiment:

Non-spiked strawberry: A strawberry that was not spiked with E. coli culture

was washed using PBS and serially diluted to 10-6. This was spread plated onto

MacConkey agar. The purpose of this control was to see if the surface of the

strawberries contained any naturally occurring coliforms. No growth was

found.

Media control: A media control was conducted to examine if the media was

selective in growing the E. coli. A pure culture of E. coli was streaked onto a

MacConkey agar plate to ensure that the media supported the growth of the

organism, and to avoid false negatives. E. coli was found to grow on the

MacConkey agar, which confirmed that the media did support the growth of

the coliforms.

PBS control: The sterile PBS was spread plated onto the MacConkey media to

ensure that it is sterile and free from contamination. If the PBS is not sterile it

can give off false positive results so it is important to check the diluent for

contamination. No contamination was present.

Experimental Results:

Sample 1 count: N – TNTC10-1 - TNTC10-2 - TNTC

Sample 2 count:N – TNTC10-1 - TNTC10-2 - TNTC10-3 - TFTC10-4 - TFTC

Sample 3 count:N – TNTC10-1 - TNTC10-2 - 17910-3 - 16410-4 - TFTC

10-3 - 6310-4 - TFTC

Discussion of results:

It was decided after conducting this experiment that there were too many variables

and the experimental design should be changed. Initially, 1ml of the overnight culture

was pipetted onto the surface of the strawberries. However, a flaw in this

experimental design was that it was difficult to inoculate the surface area of the

berries equally and evenly using a pipette. Furthermore, the berries were left to sit in a

puddle of the nutrient broth culture. This was problematic as not all the E. coli cells

were present evenly on the surface area of the strawberries and the washing of the

strawberries would not be effective in removing all the cells that were placed on the

strawberries (as high numbers of the cells would remain in the petri dish which would

mean it would not be possible to recover the correct concentration of bacteria from the

strawberries). Furthermore, the culture should have been inoculated in PBS rather

than nutrient broth as the cells would continue to grow on the strawberries when they

were left to dry. After some research from other scientific papers that conducted

similar experiments it was decided that 10 x 10 µl of the broth should be spot

inoculated onto the surface of the berry to ensure even distribution across the surface

area of the strawberries. This should be left to incubate at room temperature in a

laminar flow hood for 1 hour, and then a standardized wash using sterile PBS would

be conducted. The neat would be serial diluted to 10-6, and 0.1 ml of these dilution

sets would be spread plated onto MacConkey agar and incubated at 37’C for 24hr.

However, the more important variable of operator precision during dilutions and

plating would first have to be investigated before performing further experiments on

strawberries.


Determine the effects of serial dilution and subsequent plating on the precision of

operators.

Why?

An experiment was designed to test the precision of serial dilution and spread plating

on inter-operator procedure and intra-operator procedure. The purpose of this

experiment was to find the difference in the precision of spread plating and

performing serial dilutions between the different operators and also to test the

precision of the dilution sets from the same operator to examine any deviation in the

results. This experiment was also designed to examine where the greater error in

recovering the cells would be; the serial dilutions or the spread plating.

How?

Each operator performed a series of 6 serial dilutions sets from the same lab grown E.

coli culture. The first 5 sets were plated once onto MacConkey agar from 10 -2 to 10-5

to examine the precision of the operators when carrying out dilutions. The 6th set was

plated 6 times onto MacConkey agar 10-2 to 10-5 to examine the precision of operators

when plating serial dilutions.

List of Equipment for testing operator precision:

Overnight E.coli culture in nutrient broth

PBS in 9ml aliquots

MacConkey Agar

Metal spreader

Metal bowl

96% ethanol

Pipette pump

1ml pipettes

Bunsen burner


An overnight culture of E.coli was prepared by inoculating nutrient broth with

a colony of E.coli from a streaked plate.

Culture left at room temperature on laboratory bench for weekend.

After weekend, 10 ml of nutrient broth was added to revive 10ml prepared

culture from previous week, and left for 1 hour @ 37°C.

A universal neat was prepared for testing by adding 10ml of revived overnight

culture to 90ml of PBS and performing standard shake.

10ml aliquots were taken from the above neat for each operator to carry out

serial dilutions.

6 sets of serial dilutions were carried out by each operator from neat to 10-5.

5 of these dilution sets had 0.1ml plated out once onto MacConkey Agar for

10-2 -10-5.

The 6th set of dilutions had 0.1ml plated onto 6 sets of MacConkey agar plates

for 10-2 -10-5.

All plates incubated @ 37°C for 24 hours.

Controls:

No controls were deemed necessary for this experiment as all operators were

working from the same batch of media and PBS and neat lab prepared sample,

so any contamination should still result in the operators obtaining the same

concentration results from their dilutions (given the dilutions were carried out

successfully).


Individual set results:

Replicate set results:

Discussion of Results:

The standard deviations for our individual set platings ranged from 12.629 to 21.719,

whereas the standard deviations for our replicate set platings were much closer (4.167

to 5.02). Based on the results of this experiment it was found that the deviation

between operators was not significant. The results between the replicates from

individual operators were also not significant. It was also found that there was a

greater error in performing the serial dilutions compared to the plating of the dilutions

between each operator and also between the replicates conducted by the same

operator. This was to be expected. These results clearly illustrate there is a larger

discrepancy between results obtained by operators when carrying out multiple sets of

serial dilutions, than there is when carrying out numerous sets of platings from one set

of serial dilutions. For our next experiment we decided to repeat the above procedure,

this time using an incurred water sample from a local canal to see if a similar pattern

of results could be obtained from a non-laboratory grown sample.


Determine the difference in precision from dilution and plating of lab grown culture

and native bacteria from an environmental source.

Why?

It was decided to conduct an experiment to test the precision of serial dilution and

spread plating of the operator using an environmental sample with (presumptive)

E.coli. The aim of this experiment was to analyze the difference in diluting and

plating environmental cultures of cells in comparison to using pure lab cultures (and if

any apparent differences in precision can be seen or attributed to the use of

environmental samples instead of pure lab cultures).

How?

We first collected water samples from the Claddagh basin, which we plated to

examine the presence of naturally occurring bacteria for use in our experiment.

However no bacteria were found, so new samples were gathered from the middle and

bank of the canal by Wards shop. These were plated also but no growth was found, so

these samples were incurred. Each operator performed a series of 6 serial dilutions

sets from the same incurred water samples. The first 5 sets were plated once onto

MacConkey agar from 10-2 to 10-5 to examine the precision of the operators when

carrying out dilutions. The 6th set was plated 6 times onto MacConkey agar 10-2 to 10-5

to examine the precision of operators when plating serial dilutions.

List of equipment for the confirmatory test:

The collection of the water samples:

Sterile containers

Gloves

Body of water (claddagh basin)

For the experiment:

Macconkey agar plates

Water sample

1ml Pipettes

Pipette pump

Bunsen burner

Metal spreader

96% ethanol in metal bowl


3 samples of water were gathered from the Claddagh basin from 3 different

locations. An experiment was carried out to test for the presence of coliforms

in each of the water samples. They were carried out in duplicate on

MacConkey agar.

The plates were incubated inverted at 44.5°C for 24 hours.

Based on the results of these, dilution sets were to be carried out to get a

quantitative measure of the amount of coliforms present at each of the sites.

However, when the results were read, there was no growth on half of the

plates, and the growth that was recorded was only 1-3 colonies per plate.

The agar plates had dried out significantly incubated at this temperature due to

improperly protecting the plates against evaporation.

The next step was gathering new samples of water from the canal by Wards

Shop. 2 different samples were taken, from the middle of the canal and by the

bank of the water.

1 dilution set was carried out on aliquots of each of the samples, which were

diluted down to 10-8.

Neat to 10-2 were plated onto Tryptone Soya Agar and 10-3 to 10-8 were plated

onto Nutrient Agar. The first dilutions were plated onto TSA because the agar

plates had been stored for some time so were predicted to give less accurate

results. However, with the expected high number of colonies at these

concentrations, it was deemed not to have too significant an impact.

These plates were incubated at 37°C for 24 hours.

A colony obtained from our previous water samples was grown up in 10ml of

Nutrient broth and incubated overnight at 37°C. This was for preparing an

incurred sample of water, for the eventuality that there were insufficient

bacteria in the water samples being tested.

The results were recorded the following day. There was no growth on the

plates, aside from 1 colony on plates at 10-7 and 10-8, which were determined to

be contamination.

1ml of the bacteria grown up was added to 80ml of each sample and to 1 L of

the same samples. This was done so that we had two different starting

concentrations to choose from for our experimentation. The water from the

middle of the canal was stored in a sealed box at room temperature to grow

over the weekend. The samples from the bank of the canal were stored in the

fridge and left to grow up over the weekend also.

After the weekend, aliquots of the litre of incurred bank water sample from the

bank were diluted out in 6 dilution sets each, from neat to 10-5. 5 of these sets

were plated out once, while the 6th set was plated out 6 times, identically to the

previous experiment.

The dilutions were plated out onto MacConkey agar.

The plates were incubated at 37°C for 24 hours.

We then repeated the dilution steps the following week using the older sample

from the middle of the canal to compare results.

Controls:

Again, no controls were deemed necessary for this experiment, as all operators

were working from the same batch of media and PBS and neat incurred water

samples, so any contamination should still result in the operators obtaining the

same concentration results from their dilutions (given the dilutions were

carried out successfully).


Individual set results (bank sample):

Replicate set results (bank sample):

Individual set results (middle sample):

No countable results were obtained due to absence of growth.

Replicate set results (middle sample):

No countable results were obtained due to absence of growth.

Discussion of Results:

The standard deviations for our individual dilution set platings of our bank sample

ranged from 10.472 to 46.069, whereas the standard deviations for our replicate set

platings of the bank sample were closer again (13.164 to 24.495). These standard

deviations for both the individual sets and the replicates were expected to be higher

than the results from the previous experiment as an environmental sample would be

more unpredictable when compared to the pure lab culture. However we can still see a

large discrepancy between results obtained by operators when carrying out multiple

sets of serial dilutions, when compared to the results obtained from the replicate

platings.

Results could not be calculated from our experiment using the sample from the

middle of the canal as there was no growth on the majority of the plates (with only 3

or less colonies appearing on 6 plates). This lack of growth could be attributed to a

number of factors:

The middle sample was two days older than the bank sample when testing was

carried out. The additional storage time could have contributed to die-off of

the majority of the bacterial cells present.

The cells were kept in the dark in a cabinet, whereas they would have been

exposed to light in the canal as they were collected from the top of the water.

This lack of exposure to light may have attributed to cell death and/or

inhibition of growth.

The middle sample was stored at room temperature, whereas the bank samples

(and the water in the river) were at colder temperatures.

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MRA Lab Report