Safety in the lab
RULES, REGULATIONS AND CODE OF CONDUCT FOR SAFETY IN THE
MICROBIOLOGY LABORATORY

APROPRIATE PROTECTIVE CLOTHING MUST
BE WORN IN THE
LABORATORY AT ALL TIMES.

SAFETY GLASSES TO BE WORN AT ALL TIMES.

(LABORATORY COATS MUST BE WORN AT ALL TIMES AND MUST BE
CLEAN AND FREE OF GRAFFITTI.)

BEHAVIOUR IN THE LABORATORY MUST BE APPROPRIATE TO REFLECT
SAFETY STANDARDS. (Performance and behaviour in the laboratory are taken
into account for CA marks.)

EATING, DRINKING AND SMOKING ARE NOT PERMITTED IN THE
LABORATORY.

HANDS MUST BE WASHED WITH SOAP ON ENTERING THE LABORATORY
AND AT ALL TIMES LEAVING THE LABORATORY.

BENCH TOPS MUST BE SWABBED WITH DISINFECTANT AT THE START
AND END OF EACH CLASS. (ETHANOL IS PROVIDED)

WASTE DISPOSAL BAGS ARE PROVIDED FOR PETRI DISHES AND OTHER
DISPOSABLES WHICH REQUIRE AUTOCLAVING.

WASTE DISPOSAL BINS ARE PROVIDED FOR WASTE PAPER .

DISCARD JARS ON THE BENCH TOPS CONTAINING DISINFECTANT ARE
PROVIDED FOR DISPOSAL OF GLASS SLIDES AND USED PIPETTES AND
PIPETTE TIPS

SINKS MUST NOT BE USED FOR WASTE DISPOSAL.

HANDLE ALL CULTURES AS IF POTENTIALLY PATHOGENIC (i.e
DANGEROUS DISEASE CAUSING ORGANISMS).

HANDLE ALL MATERIAL I.E, WATER FROM RIVERS/LAKES etc., SOIL,
SLUDGES AND MATERIALS FROM OTHER SOURCES AS CONTAINING
POTENTIAL PATHOGENS.

DO NOT LICK LABELS, PENCILS, FINGERS etc.

TRY TO PREVENT RUBBING YOUR EYES AND LIPS, BE AWARE OF THE
POSSIBILITY OF CONTAMINATION AT ALL TIMES.
 THINK ASEPTIC TECHNIQUE AT ALL TIMES

IN CASE OF ACCIDENT (BREAKAGES, SPILLAGES etc.) INFORM THE
LECTURER IMMEDIATELY.

ALWAYS LEAVE THE LABORATORY CLEAN AND TIDY FOR YOUR NEXT
CLASS. Clean bench top of stains and put away microscopes, hot plates etc.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Water Microbiology
The quality of water, for both drinking and recreation
purposes, is now a matter of national and International
concern. The European Commission has issued a council
directive relating to the quality of water supplies (The
Drinking Water Directive (80/778/EEC), 1980). A more
recent Directive relates to the quality of water intended for
human consumption (98/83/EC)
The Irish Government brought the original directive into law
by introducing the European Communities (Quality Of Water
Intended For Human Consumption) Regulations,1988 which
are the statutory basis for protection of drinking water quality
in Ireland. The bodies charged with the implementation of
the regulations are the sanitary authorities, which then
furnish the results to the EPA in order to publish the annual
report on drinking water quality.
Officially approved methods for the bacteriological
examination of water are given by the UK Department of
Health (DHSS, 1985) and in the USA by the American
Public Health Association (APHA, 1986).
In relation to public health the principal tests applied to water
are:o the viable plate count, and those for
o coliform bacteria,
o faecal coliform (E.coli),
o faecal enterococci and
o sulphite-reducing Clostridia.
The viable plate count is carried out at 22C and 37C.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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The terms used for the microorganisms may be defined as
follows:
Coliform bacteria are members of the Enterobacteriaceae and
include the genera Citrobacter, Enterobacter,
Escherichia, Hafnia, Klebsiella and Serratia.
These grow at 37°C and possess a -galactosidase
enzyme.
Faecal coli, also known as thermotolerant coli
refers to Escherichia coli, which grows and
produces indole at 44.5°C.
Faecal enterococci are members of the genus Enterococcus,
and include E. faecalis, E. faecium and E. durans and
belong to the family Streptococcaceae





They grow at 10°C and 45°C, in the
presence of 40% bile,
6% NaCI, and on
standard azide media, and
hydrolyse aesculin.
Sulphite-Reducing Clostridia refer to Clostridium
perfringens.
These bacteria are
Gram positive rod shaped
 anaerobic,
 produce spores and
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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 reduce sulphite, blackening the medium
which is characteristic and cause
 stormey clot in litmus milk
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Sampling.
Water samples are usually collected using sterile 300 ml or
500 ml bottles supplied by the laboratory.
Plastic is replacing glass bottles because of concern about
glass in food preparation and recreational areas.
For samples of chlorinated water the bottles must contain
sodium thiosulphate (0.1 ml of a 1.8% solution per 100 ml
capacity) to neutralize chlorine.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Viable plate counts
These are required under EU Directives.
Nutrient agar (Yeast extract agar) is used and tests are done
in duplicate with undiluted and serially diluted samples. One
set is incubated at 20-22°C for 3 days and the other at 37°C
for 24-48 hours.
Procedure:
Prepare serial dilutions of sample from 100,10-1,10-2,10-3,
using the diluent. (Use either Ringers or Peptone water).
Observe aseptic technique throughout.
Label two series of petri dishes, one for 22C and 37C in
duplicate.
Starting with 10-3 dil. carry out plate count using the pour
plate technique and carry on with 10-2,10-1,100.
Pipette 1 ml of sample into petri dishs in duplicate and add
molten ager.
Mix agar and sample very carefully to disperse the bacteria.
NB(You have only one chance to do this as you cannot go
back to undo the solid agar)
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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After incubation count the colonies carefully and calculate
the number of CFU's per ml of the original sample,
using the dilution factors.
Prepare a table showing the results.
***Why incubate at 37C ?***
The target values are
CFU/ml at 37°C.
<100
CFU/ml at 22°C and
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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<
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Presumptive Coliform test:
MPN method with MacConkey broth.
Multiple Tube Method to determine Most Probable Number
of Coliforms.
Select the range according to the expected purity of
the water:
Mains chlorinated water
A and B
Piped water, not chlorinated A, B and C
Deep well or borehole
A, B and C
Shallow well
B, C and D
No information
A, B, C and D
A: 50 ml of water to 50 ml of double-strength broth.
B: 10 ml of water to each of five tubes of 10 ml of doublestrength broth.
C: 1 ml of water to each of five tubes of 10 ml of singlestrength broth.
D: 0.1 ml of water to each of five tubes of 10 ml of singlestrength broth.
Question.
Why are sample volumes C and D
usually omitted when sampling treated
mains water?
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Procedure:
You have on your bench in a test tube rack the series:
five tubes of 10 ml of double-strength broth.
Add 10 ml of sample to each.
five tubes of 10 ml of single-strength broth.
Add 1 ml of sample to each.
five tubes of 10 ml of single-strength broth.
Add 0.1 ml of sample to each.
*************************************************
Incubate at 35-37°C and note the numbers of tubes showing
acid and gas after 48 h.
Tap any tubes showing no gas. A bubble may then form in
the Durham's tube.
Consult the MPN tables and read the most
probable number of presumptive coliform
bacilli/100 ml of water. Report the results.
Small amounts of gas occurring after 48 h in presumptive
tubes are disregarded unless the presence of coliform
bacilli is confirmed by plating.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Confirmatory test.
From each tube showing acid and gas, inoculate a tube of
MacConkey broth and a tube of peptone water.
****Incubate these at 44.5°C for 24 h in a reliable water-bath
(Eijkman test) along with controls of known strains of
E. coli (which grows at 44.5°C) and K. aerogenes
(which does not).
Plate also from positive tubes on MacConkey agar, Eosine
Methylene Blue (EMB) and nutrient agar.
 Observe gas formation at 44.5°C and test the peptonewater culture for indole.
 Do Gram stain and oxidase test on growth from nutrient
agar.
Should find Gram negative, non spore
forming and oxidase negative cultures.
Only E. coli produces acid, gas and indole at 44.5°C.
Read the most probable numbers of E.
coli ('faecal coliform') from Tables and
report the results.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Questions.
What are the components of the MacConkey broth?
What is the Carbon and energy source in the
medium?
How does this medium encourage the growth of
coliforms?
What is the gas in the Durham tube composed of
and where does it come from?
Remember that the organisms cultured from any positive
37°C tube and grown at 44°C represent coliforms cultured
from the volume of water placed in the 37°C tube.
For example:
Tubes positive at 37 °C
50 ml
1
10 m1
2
1 ml MPN/100 ml
2
10
presumptive coli
Tubes positive at 44.5 °C
1
1
0
3 ..E. coli
For further investigation, subculture colonies from the
MacConkey/EMB plate for biochemical tests, e.g. with an
API kit or the IMViC test.
The IMViC (I = indole, M = methyl red, V = Voges-Proskauer, C = Citrate)
tests are not routinely used in water microbiology.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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***Acid and gas in MacConkey broth, may occasionally be
due to spore formers. e.g. Cl. perfringens at both 37°C and
44°C.
Question.
However, these organisms do not
grow on the MacConkey or EMB
plates.
Explain why?
Most raw waters showing acid and gas do in fact contain
coliform bacilli, but in about 5% of chlorinated waters acid
and gas are caused by C. perfringens.
The target levels for coliforms and E.coli are absence
from 100 ml.
IMViC test
I = indole production from tryptophan,
M = methyl red, indicates acid production from glucose,
V = Voges-Proskauer, indicates neutral end products from glucose i.e. Acetyl
methyl carbinol,
C = Citrate utilization by the suspect culture
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Coliform test: membrane filter method
Advantages of using membrane filter techniques for
waters
(1) Speed of obtaining results.
(2) Saving of labour, media, glass and cost of materials if the
filter is washed and re-used.
(3) Sample can be filtered on site, if the filter is placed on
transport medium and posted to the laboratory, thus avoiding
delay in transporting the sample.
(4) Organisms can very easily be exposed to pre-enrichment
media for a short time at an advantageous temperature.
Disadvantages of using membrane filter techniques for
waters
(l)There is no indication of gas production (some waters
contain large numbers of non-gas producing lactose
fermenters capable of growth in the medium).
(2)Membrane filtration is unsuitable for waters with high
turbidity and low counts because the filter will become
blocked before sufficient water can pass through it and
(3)Large numbers of non-coliform organisms capable of
growing on the medium may interfere with coliform growth.
If large numbers of water samples are to be examined and
much field work is involved the membrane method is
undoubtedly the most convenient.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Procedure:
Set up the membrane filtration unit as demonstrated.
 Prime the membrane by passing approx. 20 ml of sterile
water through.
 Pass two separate l00-ml volumes of the water sample
through 47-mm diameter membrane filters.
Question.
What is the pore size of the
membrane you are using?
****If the supply is known or is expected to contain more
than l00 coliform bacilli/l00 ml, use l0 ml of water
diluted with 90 ml of quarter-strength Ringer's solution.
 Place sterile absorbent pads in sterile petri dishes and
pipette 2.5-3 ml of m Endo broth over the surface and
allow to absorb.
 Place a membrane face up on each pad.
 Incubate one membrane at 44.5C and one at 37C for
48h.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Counting and reporting results.
Count the typical colonies only and report
as presumptive coliform and E. coli /l00 ml
of water.
Cl. perfringes does not grow.
Completed test
 Several colonies from the membrane are subcultured into
lactose broth fermentation tubes and on a nutrient agar
slope.
Both are incubated at 35°C for 24 h.
 Gas in the broth and a Gram-negative non-sporing rod on
the slope is evidence of coliform bacilli.
Gram stain the culture and carry out the OXIDASE TEST.
For the oxidation of glucose many bacteria
utilize a respiratory transport chain, a collection
of cytochromes and other enzymes terminating
in cytochromes oxidase. Bacteria producing
cytochromes oxidase can oxidase the substrate
tetramethyl- para-phenylene diamine
hydrochloride, the oxidase reagent, which is
oxidised to produce an intensely coloured purple
product in about 10 seconds.
 Follow the instructions of your demonstrator when
carrying out the Oxidase test.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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4-Methyl umbelliferyl-ß-D-gluconate (MUG)
may be added to the tryptone water to give an
additional test for, ß-glucuronidase activity
which is positive only for E. coli (ca. 90% of
strains) and some shigellas.
MUG is hydrolysed to give a fluorescent
compound, detected by exposure to UV light.
The indole reagent may then be added.
Questions
How many bacteria were in the water sample?
How many of these bacteria were total
coliforms?
How many were faecal coliforms?
Explain the significance of these results.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Faecal Enterococci in water
These organisms are useful indicators when doubtful results
are obtained in the coliform test. They are more resistant than
E. coli to chlorine and are therefore useful when testing
repaired mains. Group D streptococci only are significant.
This group of microorganisms were known as faecal
Streptococci, but are now referred to as Enterococci.
MPN method.
Use one of the azide broths, e.g. azide glucose broth or
Enterococcus Presumptive Broth.
 Add 50 ml of water to 50 ml of double-strength medium.
 Add 10 ml of water to each of five tubes of 10 ml of
double-strength broth.
 Add 1 ml of water to each of five tubes of 5 ml of singlestrength broth.
 Incubate at 37°C for 48/72 h.
 Subculture any tubes showing acid production to tubes of
single-strength medium and incubate at 44.5°C for 18/24
h.
Record tubes showing acid and
consult the MPN tables.
Present the results in a table.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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 Subculture each presumptive positive tube to ethyl
violet azide broth and incubate at 44.5°C for 24-48 h.
Turbidity and a purple-stained button of growth at the
bottom of the tube indicate enterococci.
Confirm by microscopic examination for short-chain
streptococci.
Report your results in a table.
Questions
What are the components of the
medium used in the MPN method?
Why is azide used in the medium,
give an explanation?
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Membrane method
 Pass l00 ml of water through a membrane filter
previously primed with sterile water, and place the filter
on a plate of membrane enterococcus agar.
 Incubate at 44.5°C for 48/72 h.
 All red or maroon colonies are presumptive positives.
 Carefully remove the filter and place colony-side down
onto a plate of aesculin bile agar to imprint the colonies.
 Incubate at 37°C for 12 h. A black zone appears under
colonies of faecal enterococci.
Carry out the catalase test.
The target level for faecal enterococci is
absence from 100 ml.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Sulphite-reducing clostridia
MPN method
Heat the sample to 75°C in a water-bath and hold it at this
temperature for 10 min.
Culture as follows in Differential Reinforced Clostridia
medium.
Add 50 ml of the sample to a 50 ml bottle of double strength
medium,
10 ml to each of five 10 ml tubes of double strength medium,
and
1 ml to each of five l0 ml tubes of single strength medium.
Overlay each medium with sterile mineral oil (2 cm deep) to
exclude as much air as possible.
Questions.
Why is the water sample preheated before
the analysis is carried out?
Why are the samples overlayed with oil,
explain the reason?
What makes the medium differential for
Clostridium?
Explain the black stain in the medium.
Cap and incubate at 37°C for 48h.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Tubes showing blackening are presumptive positives but
other clostridia may give this reaction.
Confirm by subculture in litmus milk medium.
Incubate at 37°C overnight and record tubes showing stormy
clot fermentation.
Q Explain what the stormy clot reaction
is?
 Carry out a spore stain.
Consult the MPN tables and report
results in table form.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Membrane method.
Prime the filter in the usual manner.
Pass 100 ml of the heated sample through a 47 mm, 0.45 µm
filter.
Place the filter face downwards on the surface of Bismuth
Sulphite agar.
Pour 20 ml of the same medium, cooled to 50°C, over the
surface and when this has solidified incubate the plates at
44°C anaerobically.
Count the black colonies with haloes. These are probably
Clostridium perfringens.
If too many are present all the medium will be blackened.
****The target levels for Cl. perfringes
are < 20 per ml.
Report results in your manual.
What are the ingredients of the Bismuth
Sulphite agar?
MPN Tables here
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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HYGIENE MICROBIOLOGY.
Assessing Microbiological Quality: Personal Hygiene,
Surfaces, Air and Product/Materials.
Personal Hygiene
A simple method of assessing Bacteriological quality of hands (an
indication of personal hygiene) is to take contact finger prints on the
surface of agar plates.
This may be used to evaluate the efficiency of hand washing procedures,
or to evaluate the effictiveness of disinfectants and hand wash solutions.
Method:
Agar Medium: TSA, Mannitol Salt, McConkey.
Clearly mark out on the back of the agar plate the areas onto which the
fingers are to be placed.
Take Two agar plates.
Label one plate ‘before washing’ and the second one ‘after washing’.
Carefully imprint each finger onto the agar plate, maintaining contact for
three seconds.
Wash your hands and dry them and repeat for the same hand on the
second plate.
Label and incubate @ 37C-24/48 hrs.
After incubation, report on the numbers of CFU’s per hand and assess the
effect of hand washing.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Surfaces.
The bacteriological quality of surfaces can be assessed by using agar
contact plates (Rodac plates) or by using a swabbing technique.
Contact plates.
Contact plates are poured using the molten agar supplied.
TSA, Mannitol salt, McConkey agar and Sabaroud Dextrose agar.
13 ml of molten agar is carefully poured into the agar plate and allowed
to set.
The agar plates are used to take an imprint of the surface under
examination, incubated @ the appropriate temperature and examined.
Report your results.
Swabs
Templates outlining an area of 5 cm2 are first sterilized.
The template is placed into position and the area within the template is
washed with a sterile cotton wool swab moistened with some sterile
diluent.
The swab is broken off into the diluent.
A dry swab is now used to wash the area within the template and is also
placed into the diluent.
The diluent is shaken for 3 minutes.
A 1/10 dilution is carried out and the number of microorganisms is
determined by the pour plate method using TSA.
Plates are incubated @ 32C for 48 hrs.
After incubation examine the plates and record your results.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Air Analysis.
Some of the devices and methods used in the bacteriological analysis of
air are as follows:Casella Slit–to–agar sampler;
Anderson Two Stage Sampler;
Biotest Centrifugal Air Sampler;
Hawksley Filter
Surface Air Sampler (SAS)
Settle Plates.
The Casella Slit–to–agar sampler is set up as demonstrated.
Air is sampled through the slits and impacted onto the surface of a plate
of TSA to collect bacteria and Sabaroud Dextrose Agar to collect yeasts
and moulds.
After incubation at the appropriate temperatures, CFU’s are counted and
reported as CFU’s per m3 air sampled.
Q? What temperature should you incubate to recover:
Bacteria_____________
Yeasts/moulds_____________
Table showing flow rate and volume of air sampled using various
slits in the Casella sample.
No. of slits
Flow/Min (litres)
Time of one cycle in Volume sampled
Min.
(litres)
1
175
2
350
3
525
4
700
0.5
2
5
0.5
2
5
0.5
2
5
0.5
2
5
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
87.5
350
875
175
700
1750
262.5
1050
2625
350
1400
3500
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Anderson Two Stage Sampler
The instrument is set up as demonstrated.
Particles carrying microorganisms are impacted onto the surface of agar
media and incubated to allow growth to occur.
The upper chamber collects all the non respirable particles (>8.0µm ) and
the lower chamber collects respirable particles (around 4 m diam.).
The pump maintains a flow rate of 28.3 liters/min.
Use two plates of
TSA
to recover _________________incubate
________hrs
at
what
_____ºC
for
Mannitol Salt agar
to recover __________________ incubate at what _____ºC for
________hrs
Sabaroud Dextrose agar to recover ________________________
incubate at what ºC for ________hrs
Sample the air for four minutes.
Determine volume of air sampled in m3.________________
After incubation report your results as CFU’s per m3 of air sampled.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Biotest Centrifugal Air Sampler
The agar strips are carefully inserted into the device as demonstrated.
The sampler is placed into position and turned on for 4mins.
After sampling the strips are placed into their plastic containers and
labelled and incubated as appropriate.
Agar strips contain agar to recover bacteria and Yeasts and moulds.
Count the colonies on the agar strip after incubation and calculate as
follows:
The number of organisms per unit of air volume can be calculated as
follows:CFU/m3 = Colonies on the agar strip x 25
Sampling time (mins)
Settle Plates.
This method allows particle carrying microorganisms to sediment out
onto the surface of an open petri dish.
Open the lids of agar media to the air and close lids after 10 mins and 30
mins and 60 mins.
Carry out determination in triplicate.
Alternative groups in the class can use TSA or Sabaroud Dextrose Agar.
Colonies develop on the agar surface during incubation.
Count the colonies and express your results as CFU/area/time sampled.
Hawksley Filter
This system collects particles from the air onto a membrane filter. The
membrane filter is then placed onto an agar medium to collect Total
bacteria or Yeasts/moulds.
What agar would you use to collect total bacteria?__________________
What
agar
would
you
use
to
collect
yeasts/moulds?__________________
What
agar
would
you
use
to
collect
Staphylococci?__________________
Sample for 10 minutes setting the pump at 30l/min.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Calculate the volume of air sampled in m3_______________________
Express you results after incubation as CFU’s/m3 air.
SAS Surface – Air – sampler.
This unit collects particles carrying microorganisms by impaction onto
the surface of agar medium in regular contact (RODAC) plates.
The agar medium can be selected to recover any group of
microorganisms.
In this practical use TSA to recover bacteria,
Sabaroud Dextrose agar to recover Yeasts/moulds,
Mannitol Salt to Recover Presumptive Staphylococci.
Sample 1000 l of air.
Incubate the agar plates at the appropriate temperatures.
After incubation report on the number of microorganisms recovered per
m3 air.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Estimation of bioburden on products.
In order to estimate the extent of contamination on products the article in
question must be rinsed in diluent and the numbers of CFU’s determined
either by pour plate method or membrane filtration.
Method.
Preparation of product for bioburden is usually carried out in the laminar
flow safety hood.
A sample of product is may be chopped, using a sterile scissors, and the
pieces placed in one litre of diluent.
Usually three pieces of product are assayed and the result is expressed as
the mean per one item of product.
The diluent is shaken for 15 mins to dislodge attached microorganisms.
A. Pass one aliquot of 250 ml of diluent through a membrane using a
sterile membrane filtration apparatus.
Place the membrane carefully, grid side up, onto the surface of a TSA
plate.
Label and incubate aerobically @ 37C for 48 hrs.
Express your result as CFU/litre of diluent i.e. per amount of product
in the diluent or per individual product.
B. Pass a second aliquot of 250 ml of diluent through a membrane using
a sterile membrane filtration apparatus.
Place the membrane carefully, grid side up, onto the surface of a TSA
plate.
Label and incubate anaerobically in a gas jar @ 37C for 48/72 hrs.
C. Pass a third aliquot of 250 ml of diluent through a membrane using a
sterile membrane filtration apparatus.
Place the membrane onto the surface of a Sabaroud Dextrose agar
plate.
Label and incubate aerobically @ 25C for 5 days.
After incubation, count all colonies appearing on the membranes and
express the bioburden as CFU’s per product.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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With the remaining diluent carry out a plate count using the same agar
media.
Prepare a 1:10 dilution of the diluent using 9 ml Ringers.
In triplicate add 1 ml of the original sample and the 1:10 dilution to three
agar plates.
Carefully add molten TSA, swirl, allow to set and incubate @ 37ºC for
48 hrs.
In triplicate add 1 ml of the original sample and the 1:10 dilution to three
agar plates.
Carefully add molten TSA, swirl, allow to set and incubate @ 37ºC in
the gas jar for 48/42 hrs.
In triplicate add 1 ml of the original sample and the 1:10 dilution to three
agar plates.
Carefully add molten Saboraud Dextrose agar, swirl, allow to set and
incubate @ 25ºC for 5 days.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Results:
Air Analysis:
Biotest
Filter
SAS
Casella
Anderson
CFU /m3
CFU /m3
CFU /m3
CFU /m3
CFU /m3
Anderson
%
non %
respirable respirable
Total bacteria
Yeasts/moulds
Mannitol
fermenters
Total
Microorganisms
Swabs:
determine the swabbed area.
CFU/ (
) cm2
coliforms
Surface swabbed
Total bacteria
Contact plates.
determine the contact area.
CFU/ cm2
Surface contacted
Total bacteria
mannitol
fermenters
coliforms
yeasts and moulds
Comment on results:
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Personal Hygiene.
CFU per hand
Name
before wash
after wash
Comment on results:
Bioburden on product
CFU/product
bacteria
description of product
Aerobic
Anaerobic
yeasts/moulds
Comment on results:
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Microbiological Analysis of Soils and Sediments.
In these practicals we will analyse soils and sediments for a variety of microbial
populations in order to get some idea of the diversity of microorganisms present in
these environments. It is possible to further analyse these soils to discover the
functions that these microbial populations are responsible for in their natural habitat,
i.e. recycling carbon, nitrogen, sulphur and phosphorus as well as production of
organic acids and gasses and mobilization of metals and microbial corrosion.
Degradation or detoxification of a wide variety of toxic organic chemicals including
hydrocarbons; aliphatic, aromatic and halogenated, are also carried out by these
populations.
Soils are a complex and heterogenous environment containing many discontinuous
microhabitats, and therefore presents a difficult challange to the investigator.
In the first practical determine the following:Microbial Population
Method
Medium
psychrophiles
mesophiles
thermophiles
spread plate
Pour plate
Pour plate
peptone yeast extract agar.
peptone yeast extract agar.
peptone yeast extract agar.
Total Fungi(Yeasts/Moulds)
Pour plate
Malt Agar(acidified)
Actinomycetes
Pour plate
Actinomycete agar
Total Bacterial Numbers
Method:
• Carefully weigh out 10 g of soil and add to 90 ml diluent in wide necked flaskes.
• Mix by gentle shaking for 5 mins. to disperse microorganisms into suspension.
Allow heavy particulates to settle out. This is the 10-1 dilution.
• Now carry out serial dilution in 90 ml diluents to 10-6.
•
•
•
For the bacteria, using the pour plate technique, add 1 ml of sample in duplicate
from 10-6, 10-5, 10-4, 10-3 to eight petri dishes labelled for 22C, repeat for 32C
and 55C. Add cooled, molten peptone yeast extract agar, mix carefully.
When completely solid, invert and incubate series at 22C, 32C and 55C,
examine regularly until no further colonies appear on the plates and note the
numbers.
For the psychrophiles, the pour plate technique cannot be used as the temperature
of the agar would kill the heat sensitive bacteria. Using the spread plate
technique, pipette 0.1 ml of suspension from 10-5, 10-4, 10-3 and 10-2 onto the
prepoured chilled agar and spread evenly with the glass spreader. Allow to dry
and incubate at 4-7C for seven days.
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•
For the fungi, using the pour plate technique, add 1 ml of sample from each
dilution 10-5, 10-4, 10-3 and 10-2 10-1 to two petri dishes. Add cooled, molten Malt
Agar (acidified pH 4.5), mix carefully. When completely solid, label, invert and
incubate pairs at 22C until the next class.
•
For the actinomycetes, using the pour plate technique, add 1 ml of sample from
each dilution 10-5, 10-4, 10-3 and 10-2 10-1 to two petri dishes. Add cooled, molten
Actinomycete agar, mix carefully. When completely solid, label, invert and
incubate pairs at 22C until the next class.
•
Report the numbers of CFU's per g dry weight of soil.
•
Determine the dry wt. of the soil using 10g of wet soil in metal trays in the oven
at 104C for 24 hrs. and dry to constant weight using a desiccator.
•
Measure the pH of the soil and record. Make a slurry of the soil (10 g) in CaCl2
solution (20 ml 0.01M CaCl2).
Media
Glycerol Caesin agar:- in 1000ml deionised water dissolve the following:- 10 g
glycerol; 0.3 g caesin; 2.0 g KNO3; 2.0 g NaCl; 2.0g K2HPO4; 0.05g
MgSO4.7H2O; 0.02 g CaCO3; 0.01 g FeSO4.7H2O; 18 g agar and 50 mg
cycloheximide. After autoclaving adjust to pH 7.0 with conc. HCl.
Malt extract agar ( Acidify to pH 4.5 with tartaric acid)
Peptone yeast extract agar:- in 1000ml deionised water dissolve the following:- 5 g
peptone, 3 g yeast extract, and 15 g agar. After autoclaving but when cool, add 10
ml 1.0M CaCl2. After autoclaving adjust to pH 7.0 with conc. HCl
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Results
CFU/g dry weight soil
Bacteria
Soil type
psychrophiles
mesophiles
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
thermophiles
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Actinomycetes Yea
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Isolation of Starch Protein and DNA degraders.
In order to demonstrate the presence of degraders form soil you can
transter colonies from the mesophile plates from the previous experiment
onto agar containing starch, protein and DNA.
Microorganisms that live in soil habitats frequently encounter substrates
in the form of polymers, and in order to extract nutrients for growth must
degrade the polymers to soluble components e.g. in the case of
carbohydrates, i.e. starch is hydrolysed by the enzyme amylase to
produce sugars.
What are the components that make up proteins and Nucleic acids?
Materials:
Each person needs one plate of:- starch agar; Casein agar and DNase
agar.
Procedure:
Using a loop aseptically transfer a portion of a colony from the agar plate
from the last practical onto the agar medium under test.
You can transfer eight-ten suspect colonies if you carefully spot the
colonies onto the plates with sufficient space between colonies. Incubate
at 22C for 48 hrs.
Include uninoculated plates as controls.
TSA agar with either
starch, casein or DNA
1
Source of colonies
from mesophile plate
2
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After incubation, examine the plates as follows:For starch degraders, flood the plate with Iodine solution and and leave
for a few minutes to allow the starch to react with the iodine. Starch
degradation is revealed by clear zones surrounding the degrading
colonies.
For protein degraders, a clear zone around any colony indicates protein
degradation.
For DNA degraders, flood the plate with 1M HCl and leave to develop.
HCl precipitates DNA in the agar leaving clear zones surrounding
colonies with the ability to degrade DNA.
Report your results.
What proportion of the colonies degrade the polymes, and do the same
colonies degrade all the polymers?
Record your observations.
If time permits carry out a Gram stain on the colonies.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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ESTIMATION OF MICROBIAL ACTIVITY BY FLUORSCEIN
DIACATATE HYDROLYSIS
Spectrophotometric determination of the hydrolysis of fluorescein
diacetate (FDA) to fluorscein can be used as a sensitive and rapid
method for determining microbial activity in soil. FDA is hydrolyzed by
a variety of enzymes i.e. proteases, lipases and esterses to fluorscein and
changes in fluorscein can be followed by measuring the absorbance at
490 nm.
METHOD.
FDA is dissolved in acetone 2mg/ml and stored as a stock solution (at 20°C).
Replicate samples of soil (10 g) are placed in conical flasks with 20 ml
sterile sodium phosphate buffer 60 mM, (pH 7.6).
To each sample, 0.1 ml FDA is added (10 µg/ml final concentration).
The flasks are incubated on the shaker at 27°C for 1-2 hr.
The reaction is terminated by adding acetone (50% final conc.).
The samples are then centrifuged for 5 mins. followed by filtration to
clear the sample.
Absorbance is then determined by reading at 490 nm using a
colourimeter.
Autoclaved soil treated exactly the same way is used as a control blank.
Activity is expressed as Abs @ 490/hr/g dry wt soil.
Samples:
Loam soil sieved, loam soil with various additives i.e. diesel, starch,
cellulose, protein.
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ISOLATION OF BACTERIOPHAGE.
PRELIMINARY ENRICHMENT OF 'PHAGE.
Raw sewage or river water is centrifuged and the supernatant collected. The
bacteria are removed from the supernatant, either by membrane filtration or
by inactivation using chloroform (six or seven drops of chloroform are
added to 10 ml of sample. The tube is shaken to ensure that the water is
saturated with the solvent. The chloroform is allowed to settle.
In water samples, where the concentrations of viruses is low, it may be
necessary to concentrate the viruses by adsorbing them onto a material such
as hydroxyapatite or aluminium sulphate.
1. Add the filtered sample containing the virus to about 20 g of
hydroxyapatite in a 1 l conical flask and shake rapidly for 5 mins. Collect the
hydroxyapatite in a Buchner funnnel, and discard the filtrate. The viruses on
the hydroxyapatite can be eluted using a dilute phosphate buffer (0.8M
Na2HPO4 22.6g/100ml (80ml) + 0.8M NaH2PO4 24.96g/200 ml (20ml)).
2. Naturally occurring coliphages can be concentrated according to the
following procedure:
2 ml of a 10% Al2(SO4)3 solution is added to 1 litre of the sample, the pH is
adjusted to 5.5 with HCl and the sample left ovrenight at 6°C. The Al(OH)3
flocs which have formed together with the adsorbed phages are centrifuged at
3,000 g for 5 min.
The sediment is then resuspended in 10 ml 0.1 M citrate buffer with a pH of
4.7.
1 ml concentrate is mixed with 0.5 ml of an E.coli suspension containing 108
CFU/ml and with 5.0 ml soft agar. The tube containing the mixture is
thoroughly mixed and poured into a Petri dish containing 20 ml tryptone
soya yeast extract agar. The plates are counted after 24 hours incubation at
37°C.
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Brain Heart Infusion broth (double strength) (50 ml DS in 200 ml conical
flasks) is prepared and sterilised.
To this 50 ml of the water sample suspected of containing bacteriophage is
added.
After mixing well 20 ml of a young culture of E.coli (6-8 hrs old) in peptone
water is added and incubated on the shaker table for 8-10 hrs, or until a
decrease in turbidity is observed.
Prepare a control to observe the growth of E.coli under the same conditions.
The analysis is carried out in duplicate.
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ISOLATION AND ENUMERATION OF 'PHAGE.
After incubation, the bacteria are removed from a small volume of broth by
centrifugation and membrane filtration.
The numbers of bacteriophage in the enriched broth are determined by
preparing a series of decimal dilutions in 1/4 str. Ringer's solution.
Alternatively, if only the presence or absence of the bacteriophage is required to be
demonstrated, then a more rapid procedure can be carried out.
Five ml of the enriched broth are transferred to a test tube and placed in a water bath
at 56°C for 30min. This is sufficient treatment to eliminate the bacteria while keeping
the bacteriophages active. The presence of the viruses can be detected by following the
procedure below.
The numbers of 'phage in the dilutions can enumerated either by a Miles &
Misra surface drop technique to inoculate lawn cultures of E.coli or by a pour
plate technique.
Lawn cultures of E.coli can be prepared by spreading 0.1ml of a 24 hr. culture
of E.coli onto the surface of a well dried agar plate. Allow the plates to dry.
The 'phage suspension is then dropped from calibrated droppers, onto the
surface of the plate and incubated overnight at 37°C.
After incubation the presence of 'phage is shown by a clear area or several
small clear areas known as PLAQUES, where the lawn of bacteria has been
lysed by the 'phage.
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Alternative method.
The pour plate technique is carried out by inoculating 3 ml TSA (kept molten
at 50°C) with 1 ml or 0.5 ml of an overnight broth culture of the host
bacterium, mix well to distribute the cells evenly in the agar. To this is added
0.5 ml/0.2 ml of the bacteriophage dilution, again mix well.
Work quickly to prevent the agar from solidifying, preferably in a warm
environment.
Pour the molten agar onto the surface of a warmed TSA plate and incubate at 37°C
overnight. Clear areas or plaques where the bacterium has been lysed by the
'phage are counted.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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Enrichment Isolation Of Degraders Of Organic
Contaminants
In this series of practicals you are required to isolate and prepare
a pure culture of microorganism capable of growth on one of the
organic chemicals listed below.
The procedure for isolation is one of enrichment isolation in
batch culture, although sometimes continuous culture techniques
are used to isolate organisms with a low affinity to the organic
contaminant in question.
The source of the organisms for this practical is activated
sewage, although soil represents a vast reservoir for all kinds of
microorganisms.
Procedure:
1. To the 50 ml activated sewage in a 250 ml conical flask, add
the chemical under investigation as the sole carbon source
(200 ml/l).
2. Incubate on an orbital shaker at 25C for seven days.
3. After incubation, streak a sample onto TSA, for single colony
isolation, and incubate for 4 - 7 days at 22 - 25C. Examine
daily for growth.
4. Many colonies may develop on the agar medium, and all may
be treated as suspect colonies capable of degrading the
chemical under investigation as their sole carbon source.
5. Select a single colony for investigation, describe the colony
morphology, and transfer a portion into mineral salts medium
containing the chemical under investigation as the sole
carbon source.
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6. Incubate on the shaker table, examine daily and note turbidity
as evidence of growth.
7. Streak again onto TSA for single colony isolation, and
incubate for 4 - 7 days at 22 - 25C. Again examine daily for
growth.
8. Repeat steps 5,6 and 7 until you are confident that the culture
grows in the mineral medium using the selected chemical as
sole carbon source.
9. Examine the cell morphology and report Gram stain, shape
and size.
Mineral Salts Medium.
All g/l distilled water.
KH2PO4
K2HPO4
NaNO3
NH4Cl
MgSO4.7H2O
KCl
CaCl2.2H2O
FeSO4.7H2O
0.2
0.8
0.25
0.25
0.2
0.1
0.01
0.01
Check the pH 7.2.
Examples of organics that may be used:Phenol, Ethylene Glycol, Tetradecane, Hexadecane, Nitrophenol, Cresote
various herbicides and pesticides.
Check the MSDS sheets for safety data, toxicity levels and any other relevant data.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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THE NITROGEN CYCLE.
Transformations of Nitrogen compounds are carried out by a variety of
microorganisms. Many of these microorganisms occur in soils as well as
in aquatic and marine habitats. In this series of practicals you will
demonstrate the involvement of microorganisms in various phases of the
nitrogen cycle.
1. Ammonification.
Ammonification is the hydrolytic decomposition of complex nitrogenous
substances to yield ammonia (NH3), and various other end products. A
wide variety of microorganisms (bacteria and fungi) are capable of
breaking down the proteins of animal and vegetable matter.
The ammonia thus liberated is then available as a nutrient and also to
nitrifying microorganisms.
Materials. Test tubes containing 4% peptone solution.
Soil, manure, sewage, cultures of Bacillus cereus, Pseudomonas
fluorescens and Proteus vulgaris, spotting tile, Nesslers reagent and pH
paper.
Procedure. Inoculate tubes of peptone with each of the above and
incubate at 27°C. Remember to leave one tube as an uninoculated
control. Test for the presence of NH3 with Nesslers reagent on a spotting
tile.
Note the pH, record the results.
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2. NITRIFICATION.
The transformaton of NH3 to N0 3 in soils is carried out by highly
specialized aerobic, autotrophic bacteria. Nitrosomonas derives its
energy for growth by the oxidation of NH3 to N0 2, and Nitrobacter
completes the oxidation of N0 2 to N0 3. The energy yield from these
oxidations is very low and consequently the bacteria grow slowely and
their isolation takes a long time under laboratory conditions.
-
However, it is possible to demonstrate the oxidation of NH3 to N0 3 by
using enrichment shaker flask culture.
MATERIALS. 2 x 250ml conical flasks containing 100 ml of the
enrichment medium as detailed below.
NITRITE FORMATION MEDIUM
MEDIUM
NITRATE FORMATION
(NH4)2SO4.................. .1.0g
NaNO2............................0.5g
K2HPO4..........................1.0g
K2HPO4. 7H2O...............1.0g
MgSO4.7H2O..................0.5g
MgSO4. 7H2O................0.3g
FeSO4.7H2O..................0.04g
FeSO4. 7H2O................0.04g
CaCO3...............................10g
Na2CO3........................1.0g
NaCl..................................2.0g
NaCl..............................0.5g
H2O........................................1L
H2O....................................1L
pH..........................6.8-7
pH.........................6.8-7
Autoclave the flasks at 15 psig/15 mins.
Spotting tiles, pH paper and the following reagents; Nesslers,
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Sulphanilic acid, naphthalamine, diphenylamine in conc. H2S04, urea
powder, dil. H2S04 and test tubes.
PROCEDURE.
To each flask add 9.0 g of test soil. Incubate the flasks on the shaker
table at 220 r.p.m. and at 27°C.
Each flask should be tested for the presence of NH3, N0-2 and N0-3 using
the spotting tiles and the procedure as detailed below.
Present the results in a table, showing the appearance of N0 2 and N03
during the test period.
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3. DENITRIFICATION. (dissimilatory nitrate reduction).
Under anaerobic conditions many bacteria can use the oxygen in nitrites
or nitrates to oxidise reduced organic matter. Nitrate is used as the
terminal electron acceptor and is reduced eventually to nitrous oxide.
Microbial denitrification leads to a loss of inorganic nitrogen
MATERIALS Test tubes containing denitrification medium as detailed
below, soil sample, Pseudomonas fluorescence, spotting tiles, reagents
for nitrogen analysis.
Denitrification medium. NaN03,1g; Na citrate, 9.5g; K2HP04, 2.0g;
CaCl2, 0.2g; FeCl3,trace;
H20 to 1 L. Check the pH, dispense into test tubes and autoclave at 15
psig for 15 minutes.
PROCEDURE :
Inoculate four tubes of denitrification medium with the soil samples and
the culture of Pseudomonas. and incubate at 27°C. Remember to retain
one tube as an uninoculated control. Test for the presence of nitrate and
nitrite during the incubation period. Tabulate your results.
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Qualatative tests for ammonia. nitrite and nitrate.
Test for Ammonia.
Put a drop of culture solution onto a spotting tile. Add one drop of
Nessler's Reagent. A deep yellow to brown colour indicates positive for
Ammonia.
Test for Nitrite. Add one drop of Sulphanilic acid and one drop of
Naphthalamine to a drop of culture solution. A red colour indicates
positive for Nitrite.
Test for Nitrate.
Add one drop of Diphenylamine (in conc. H2S04) to a drop of the
culture solution. A blue colour inddicates positive for Nitrate. However,
Nitrite will also give a blue colour in this test, therefore to confirm the
presence of Nitrate it is necessary to destroy any Nitrite that might be
present by boiling a little of the culture solution with urea plus dil.
H2S04 in a test tube. Cool and repeat the test for Nitrate.
Dr. Michael A. Broaders Dept. Environmental Science. Sligo
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4. NITROGEN FIXATION.
Microbial nitrogen fixation results in a net input of nitrogen into the
system and is carried out by a variety of symbiotic and free living
nitrogen fixing microbes that live in the soil. Azotobacter is a highly
aerobic, free living nitrogen fixing bacterium found in soil. Azotobacter
can be isolated from soil by sprinkling a little onto the surface of an agar
medium or into a liquid enrichment medium lacking nitrogen, as shown
below.
N-FREE MANNITOL/SUCROSE AGAR.
For isolation and cultivation of Azotobacter from soil.
Mannitol ............................................................ ..10 g
or
Sucrose .............................................................. ..1 0 g
K2HP04 ............................................................ ...0.5 g
CaC03 ............................................................... ...0.5g
MgS04 .7.H2O ................................................... ...0.025g
FeS047.H2O ....................................................... ...0.025g
Na2MoO4 .......................................................... ....0.0025g
Agar .................................................................. ....1.2/1.5g
Water................................................................. ....1 L
pH .................................................. 7.2-7.4
MATERIALS. Plates of nitrogen free agar medium, soil samples and
Ringers diluent.
PROCEDURE. Sprinkle fine soil particles sparsly over the surface of the
agar plates. Incubate the agar plates at 27°C until the next practical
period and look for typical Azotobacter colonies. These should appear as
raised, moist, glistening white colonies. Examine the cell morphology by
Gram staining.
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Attempt to purify the cells by suspending a portion of a well isolated
colony in Ringers and restreaking onto the same medium.
Alternatively, N-free liquid medium can be prepared and Azotobacter
isolated and purified by repeated subculture in shake flask culture.
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ENUMERATION, ISOLATION AND CHARACTERISATION OF
MICROORGANISMS FROM SOIL.
Before begining the microbiological analysis, determine the moisture
content of the soils so that you can express the microbial count as CFU
per gramme dry weight of the soil. In the preliminary investigation you
will estimate the numbers of bacteria, fungi and actinomycetes in the soil
samples.
MATERIALS. Soil samples, 90ml diluents, sterile 10 ml and 1 ml
pipettes.
Culture media; Soil extract agar for total bacteria,
Dextrose Nitrate agar for actinomycetes,
acid PDA and Rose Bengal agar for fungi,
McConkey or Violet Red Bile Agar for coliforms.
SOIL EXTRACT AGAR.
K2HP04 ............................. 0.5g
Soil Extract
Dextrose............................ 0.1g Mix 1kg soil with 1.51 H20,
Soil Extract .......................... 11 autoclave at 15 psig/30 mins.
Agar ............................. 12/1 5g
Filter.
pH ................................. 6.8-7.0
Sterilize at 15 psig/15 mins.
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Medium for the isolation of Actinomycetes.
DEXTROSE NITRATE ACTIDIONE AGAR.
Agar --------------------- l5 g
Dextrose ---------------- l g
KH2PO4 --------------------- 0.1 g
NaN03 ------------------- 0.1 g
KCl ---------------------- O.l g
MgS04.7H20------------ O.l g
H2O ---------------------- l l
pH------------------------ 7.0
Autoclave 15 lbs / 15 mins.
Acid PDA.
To Potato Dextrose Agar add sufficient sterile H2S04 to bring the pH
to pH 4.0. Add the acid while the agar is still molten but cool and after
autoclaving.
Rose Bengal Antibiotic Agar.
Glucose
NH4NO3
Yeast extract
MgSO4.7H2O
KH2PO4
FeSO4.7H2O
Water
Agar
Rose Bengal Dye
Neomycin
10 g
1.0 g
2.0 g
0.2 g
3.0 g
0.0025 g
1 liter
15 g
0.09 g
0.1 g
Neomycin is best filter sterilized and 1 mg added to the dry Petri dish before
adding the molten agar.
PROCEDURE. Add 10 g soil to 90 ml of diluent and shake vigerously
for 10 mins to suspend the soil completely. Allow the large particulates
to settle and continue decimal dilution to 10-7.
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Prepare pour plates in duplicate using 1 ml samples of appropiate
dilutions into the selected agar media. Incubate at 25°C and examine the
plates periodically for development of the colonies. Tabulate your
results.
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Enrichment Isolation of degrades of organics suitable for
Bioremediation.
In this series of practicals you are required to isolate and prepare
a pure culture of microorganism capable of growth on one of the
organic chemicals listed below.
The procedure for isolation is one of enrichment isolation in
batch culture, although sometimes continuous culture techniques
are used to isolate organisms with a low affinity to the organic
contaminant in question.
The source of the organisms for this practical is activated
sewage, although soil represents a vast reservoir for all kinds of
microorganisms.
Procedure:
1. To the 50 ml activated sewage in a 250 ml conical flask, add
the chemical under investigation as the sole carbon source
(200 ml/l).
2. Incubate on an orbital shaker at 25C for seven days.
3. After incubation, streak a sample onto TSA, for single colony
isolation, and incubate for 4 - 7 days at 22 - 25C. Examine
daily for growth.
4. Many colonies may develop on the agar medium, and all may
be treated as suspect colonies capable of degrading the
chemical under investigation as their sole carbon source.
5. Select a single colony for investigation, describe the colony
morphology, and transfer a portion into mineral salts medium
containing the chemical under investigation as the sole
carbon source.
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6. Incubate on the shaker table, examine daily and note turbidity
as evidence of growth.
7. Streak again onto TSA for single colony isolation, and
incubate for 4 - 7 days at 22 - 25C. Again examine daily for
growth.
8. Repeat steps 5,6 and 7 until you are confident that the culture
grows in the mineral medium using the selected chemical as
sole carbon source.
9. Examine the cell morphology and report Gram stain, shape
and size.
Mineral Salts Medium.
All g/l distilled water.
KH2PO4
K2HPO4
NaNO3
NH4Cl
MgSO4.7H2O
KCl
CaCl2.2H2O
FeSO4.7H2O
0.2
0.8
0.25
0.25
0.2
0.1
0.01
0.01
Check the pH 7.2.
Examples of organics that may be used:Phenol Ethylene Glycol
Tetradecane
Nitrophenol Cresote
various herbicides and pesticides.
Hexadecane
Check the MSDS sheets for safety data, toxicity levels and any other relevant data.
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Ecology of Phototrophs: Preperation of Microcosm to examine
Microbial ecology, The Winogradsky Column.
To 20-25 g of surface sediment from a fresh water pond add 0.5 g
CaSO4, and some organic matter, in the form of shredded filter
paper/ caesin.
Put the mixture into a 500 ml measuring cylinder and fill with fresh
water.
Expose the column to natural daylight at room temperature.
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