Sem 2 Practical

8/12/2019 Sem 2 Practical

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Safety in the labRULES, 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. Broa ders De pt . Environ m e n t al Sc ience. Sli go 1 1


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Water MicrobiologyThe quality of water, for both drinking and recreation

purposes, is now a matter of national and Internationalconcern. The European Commission has issued a councildirective relating to the quality of water supplies (TheDrinking Water Directive (80/778/EEC), 1980). A morerecent Directive relates to the quality of water intended forhuman consumption (98/83/EC)The Irish Government brought the original directive into lawby introducing the European Communities (Quality Of Water

Intended For Human Consumption) Regulations,1988 whichare the statutory basis for protection of drinking water qualityin Ireland. The bodies charged with the implementation of theregulations are the sanitary authorities, which then furnish theresults to the EPA in order to publish the annual report ondrinking water quality.

Officially approved methods for the bacteriologicalexamination of water are given by the UK Department ofHealth (DHSS, 1985) and in the USA by the AmericanPublic Health Association (APHA, 1986).

In relation to public health the principal tests applied to waterare:-

the viable plate count, and those for coliform bacteria, faecal coliform (E.coli), faecal enterococci and sulphite-reducing Clostridia.

The viable plate count is carried out at 22C and 37C.



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The terms used for the microorganisms may be defined asfollows:

Coliform bacteriaare members of the Enterobacteriaceae andinclude the genera Citrobacter, Enterobacter,Escherichia, Hafnia, Klebsiella and Serratia.

These grow at 37C and possess a -galactosidaseenzyme.Faecal coli, also known as thermotolerant colirefers to Escherichia coli, which grows andproduces indole at 44.5C.

Faecal enterococci are members of the genusEnterococcus,and includeE. faecalis, E. faecium andE. durans andbelong to the family Streptococcaceae

They grow at 10C and 45C, in the

presence of 40% bile,

6% NaCI, and on

standard azide media, and

hydrolyse aesculin.

Sulphite-Reducing Clostridia refer to Clostridiumperfringens.

These bacteria areGram positive rod shaped anaerobic, produce spores and reduce sulphite, blackening the medium

which is characteristic and cause



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stormey clot in litmus milk



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Sampling.Water samples are usually collected using sterile 300 ml or500 ml bottles supplied by the laboratory.

Plastic is replacing glass bottles because of concern aboutglass in food preparation and recreational areas.For samples of chlorinated water the bottles must containsodium thiosulphate (0.1 ml of a 1.8% solution per 100 mlcapacity) to neutralize chlorine.



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Viable plate countsThese are required under EU Directives.

Nutrient agar (Yeast extract agar) is used and tests are donein duplicate with undiluted and serially diluted samples. Oneset is incubated at 20-22C for 3 days and the other at 37Cfor 24-48 hours.

Procedure:Prepare serial dilutions of sample from 100,10-1,10-2,10-3, usingthe diluent. (Use either Ringers or Peptone water).

Observe aseptic technique throughout.

Label two series of petri dishes, one for 22C and 37C induplicate.

Starting with 10-3dil. carry out plate count using the pourplate technique and carry on with 10-2,10-1,100.

Pipette 1 ml of sample into petri dishs in duplicate and addmolten ager.

Mix agar and sample very carefullyto disperse the bacteria.

NB(You have only one chance to do this as you cannot goback to undo the solid agar)



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After incubation count the colonies carefully and calculate

thenumber of CFU's perml of the original sample,using the dilution factors.

Prepare a table showing the results.

***Why incubate at 37C ?***

The target values are


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Presumptive Coliform test:MPN method with MacConkey broth.

Multiple Tube Method to determine Most Probable Numberof Coliforms.

Select the range according to the expected purity ofthe water:

Mains chlorinated water A andB

Piped water, not chlorinated A, B and CDeep well or borehole A, B and CShallow well B, C andDNoinformation A, B, C andD

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 double-

strength broth.

C: 1 ml of water to each of five tubes of 10 ml of single-strength broth.

D: 0.1 ml of water to each of five tubes of 10 ml of single-strength broth.

Question.Why are sample volumes C and Dusually omitted when sampling treatedmains water?

Dr. Michael A. Broa ders De pt . Environ m e n t al Sc ience. Sli go 8


9/60Dr. Michael A. Broa ders De pt . Environ m e n t al Sc ience. Sli go ! 9


10/60

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 35-37C and note the numbers of tubes showingacid and gas after 48 h.

Tap any tubes showing no gas. A bubble may then form inthe 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 inpresumptivetubes are disregarded unless the presence of coliformbacilli is confirmed by plating.

Dr. Michael A. Broa ders De pt . Environ m e n t al Sc ience. Sli go 1" 10


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Confirmatory test.From each tube showing acid and gas, inoculate a tube ofMacConkey broth and a tube of peptone water.

****Incubate these at 44.5C for 24 h in a reliable water-bath(Eijkman test) along with controls of known strains ofE. coli (which grows at 44.5C) and K. aerogenes(which does not).

Plate also from positive tubes on MacConkey agar, EosineMethylene Blue (EMB) and nutrient agar.

Observe gas formation at 44.5C and test the peptone-water culture for indole.

Do Gram stain and oxidase test on growth from nutrientagar.

Should find Gram negative, non sporeforming and oxidase negative cultures.

OnlyE. coli produces acid, gasand indole at 44.5C.

Read the most probable numbers of E.

coli ('faecal coliform') from Tables and

report the results.



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Questions.What are the components of the MacConkey broth?

What is the Carbon and energy source in themedium?How does this medium encourage the growth ofcoliforms?What is the gas in the Durham tube composed ofand where does it come from?

Remember that the organisms cultured from any positive37C tube and grown at 44C represent coliforms culturedfrom the volume of water placed in the 37C tube.For example:

50 ml 10 m1 1 ml MPN/100 ml

Tubes positive at 37 C 1 2 2 10presumptive coli

Tubes positive at 44.5 C 1 1 0 3 ..E. coli

For further investigation, subculture colonies from theMacConkey/EMB plate for biochemical tests, e.g. with anAPI 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.



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***Acid and gas in MacConkey broth, may occasionally bedue to spore formers. e.g. Cl. perfringensat both 37C and44C.

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 containcoliform bacilli, but in about 5% of chlorinated waters acidand gas are caused by C. perfringens.

The target levels for coliforms andE.coli are absence

from 100 ml.

IMViC testI = indole production from tryptophan,M = methyl red, indicates acid production from glucose,V = Voges-Proskauer, indicates neutral end products from glucose i.e. Acetylmethyl carbinol,C = Citrate utilization by the suspect culture



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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 thefilter is washed and re-used.

(3) Sample can be filtered on site, if the filter is placed ontransport medium and posted to the laboratory, thus avoiding

delay in transporting the sample.

(4) Organisms can very easily be exposed to pre-enrichmentmedia 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 waterscontain large numbers of non-gas producing lactosefermenters capable of growth in the medium).

(2)Membrane filtration is unsuitable for waters with highturbidity and low counts because the filter will becomeblocked before sufficient water can pass through it and

(3)Large numbers of non-coliform organisms capable ofgrowing on the medium may interfere with coliform growth.

If large numbers of water samples are to be examined andmuch field work is involved the membrane method isundoubtedly the most convenient.



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Procedure:Set up the membrane filtration unit as demonstrated.

Prime the membrane by passing approx. 20 ml of sterilewater through.

Pass two separate l00-ml volumes of the water samplethrough 47-mm diameter membrane filters.

Question.

What is thepore sizeof the

membrane you are using?

****If the supply is known or is expected to contain morethan 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 andpipette 2.5-3 ml of m Endo broth over the surface andallow to absorb.

Place amembrane face up on each pad.

Incubate one membrane at 44.5C and one at 37C for48h.



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Counting and reporting results.

Count the typical colonies only and report

aspresumptive coliform andE. coli/l00 mlof water.

Cl. perfringes does not grow.

Completed test

Several colonies from the membrane are subcultured intolactose broth fermentation tubes and on a nutrient agarslope.

Both are incubated at 35C for 24 h.

Gas in the broth and a Gram-negative non-sporing rod onthe slope is evidence of coliform bacilli.

Gram stain the culture and carry out the OXIDASE TEST.

For the oxidation of glucose many bacteriautilize a respiratory transport chain, a

collection ofcytochromesand other enzymesterminating incytochromes oxidase. Bacteria

producing cytochromes oxidase can oxidase thesubstratetetramethyl- para-phenylene diaminehydrochloride, the oxidase reagent, which isoxidised to produce an intensely colouredpurple product in about 10 seconds.

Follow the instructions of your demonstrator whencarrying out the Oxidase test.



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4-Methyl umbelliferyl--D-gluconate (MUG)

may be added to the tryptone water to give an

additional test for, -glucuronidase activitywhich 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.



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Faecal Enterococci in water

These organisms are useful indicators when doubtful resultsare obtained in the coliform test. They are more resistant thanE. coli to chlorine and are therefore useful when testingrepaired 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 orEnterococcus 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 ofdouble-strength broth.

Add 1 ml of water to each of five tubes of 5 ml of single-strength broth.

Incubate at 37C for 48/72 h.

Subculture any tubes showing acid production to tubes ofsingle-strength medium and incubate at 44.5C for 18/24 h.

Record tubes showing acid and

consult the MPN tables.

Present the results in a table.



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Subculture each presumptive positive tube to ethylviolet azide broth and incubate at 44.5C 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-chainstreptococci.

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. Broa ders De pt . Environ m e n t al Sc ience. Sli go 1! 19


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Membrane method

Pass l00 ml of water through a membrane filterpreviously primed with sterile water, and place the filter

on a plate of membrane enterococcus agar.

Incubate at 44.5C 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 37C for 12 h. A black zone appears undercolonies of faecal enterococci.

Carry out the catalase test.

The target level for faecal enterococci isabsence from 100 ml.



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Sulphite-reducing clostridia

MPN method

Heat the sample to 75C in a water-bath and hold it at thistemperature for 10 min.

Culture as follows in Differential Reinforced Clostridiamedium.Add 50 ml of the sample to a 50 ml bottle of double strengthmedium,10 ml to each of five 10 ml tubes of double strength medium,

and1 ml to each of five l0 ml tubes of single strength medium.

Overlay each medium with sterile mineral oil (2 cm deep) toexclude as much air as possible.

Questions.

Why is the water sample preheated beforethe 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 37C for 48h.



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Tubes showing blackening are presumptive positives butother clostridia may give this reaction.

Confirm by subculture in litmus milk medium.

Incubate at 37C overnight andrecord tubes showing stormyclot fermentation.

Explain what the stormy clot reaction

is?

Carry out a spore stain.

Consult the MPN tables and report

results in table form.



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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 mfilter.

Place the filter face downwards on the surface of BismuthSulphite agar.

Pour 20 ml of the same medium, cooled to 50C, over thesurface and when this has solidified incubate the plates at

44C anaerobically.

Count the black colonies with haloes. These are probablyClostridium 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



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HYGIENE MICROBIOLOGY.

Asses sin g Microbiologic al Quality: Person al

Hygiene

!ur"aces Air an# Pro#uct$Materials%

Per so n al Hygi en eA si&'le &et (o# o" as sess ing )ac te ri ologi cal *uality o"

(an #s +an in#ication o" 'erson al (ygien e, is to ta- e

contact "inger 'rints on t(e sur"ace o" agar 'lates %

.(is &ay be use# to e/alua te t(e e""iciency o" (an#

as (in g 'roc e# u r es or to e/ al ua t e t( e e""icti/ en e s s o"

#isin"ectants an# (an# as( solut ions%

Method:

Agar Me#iu&: .!A Mannitol !alt Mcon-ey%

learly &ar - out on t(e bac- o" t (e ag ar 'lat e t(e ar ea s

onto (ic( t(e "ingers are to be 'lace#%

.a-e .o agar 'lates%

abel one 'late b e or e ! as h in g " an# t(e secon# onea ter ! ash i ng" %

are"ully i&'rint eac( "inger onto t(e agar 'late

maintaining contact #or three seconds.

as( your (an #s an# #ry t(e & an# re' ea t "or t(e sa &e

(an# on t(e secon# 'la te %

abel an# incubate 3724$48 (rs%

A"te r incubat ion re'or t on t(e nu&bers o" s 'er (an#

an# assess t(e e""ec t o" (an# as(ing%



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#$ra%es ..(e bac teriological *ua li ty o" sur"aces can be assesse# by

using ag ar cont ac t 'lat es +o# ac 'lat es , or by using a

sabbing tec(ni*ue%

Conta% t &l ate s .

ontac t 'lat es a re 'oure# using t(e &ol ten agar su''li e# %

.!A Mannitol sal t Mcon-ey agar an# !abarou# e;trose

agar%

13 &l o" &ol ten agar is care"u lly 'oure# into t(e agar 'la te

an# alloe# to set%

.(e a ga r 'lat es ar e us e# to ta- e an i&'rin t o" t( e sur" ac e

un#er e;a&ination incubat e# t(e a''ro'riat ete &' e r a t u r e an # e; a&i n e# %

e'ort your resul ts%

#!abs

.e&'lates outlining an area o" 5 c& 2 are "irst sterili


26/60

'ir 'nalysi s.Some of the devices and methods used in the bacteriological analysis ofair are as follows:-Casella Slittoagar sampler;

Anderson Two Stage Sampler;Biotest Centrifugal Air Sampler;Hawksley FilterSurface Air Sampler (SAS)Settle Plates.

The Casella Slittoagar sampler is set up as demonstrated.Air is sampled through the slits and impactedonto the surface of a plateof TSA to collect bacteria and Sabaroud Dextrose Agar to collect yeasts

and moulds.After incubation at the appropriate temperatures, CFUs are counted andreported as CFUs per m3air sampled.

Q? What temperature should you incubate to recover:Bacteria_____________Yeasts/moulds_____________

Table showing flow rate and volume of air sampled using variousslits in the Casella sample.No. of slits Flow/Min (litres) Time of one cycle in

Min.

Volume sampled

(litres)1 175 0.5

25

87.5

350875

2 350 0.525

1757001750

3 525 0.525

262.510502625

4 700 0.52

5

3501400

3500

Anderson Two Stage Sampler



27/60

The instrument is set up as demonstrated.

Particles carrying microorganisms are impactedonto the surface of agar

media and incubated to allow growth to occur.The upper chamber collects all the non respirable particles (>8.0m ) 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 ofTSAto recover _________________incubate at what _____C for________hrs

Mannitol Salt agarto recover __________________ incubate at what _____C for________hrs

Sabaroud Dextrose agar to recover ________________________ incubateat what C for ________hrs

Sample the air for four minutes.

Determine volume of air sampled in m3.________________

After incubation report your results as CFUs per m3of air sampled.



28/60

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 andlabelled and incubated as appropriate.Agar strips contain agar to recover bacteria and Yeasts and moulds.

ount t(e coloni es on t(e aga r stri' a"t er incuba ti on an#

calculate as "ollos:

The number of organisms per unit of air volume can be calculated as

follows:-

CFU/m3= Colonies on the agar strip x 25Sampling 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 30mins 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.

Ha!(sle y )ilter.(is syste& collects 'articles "ro& t(e air onto a &e&brane "ilter% .(e

&e&brane "ilter is t(en 'lace# onto an agar &e#iu& to collect .otalbacteria or =easts$&oul#s%

(at agar oul# you use to collect total bacteria>

??????????????????

(at agar oul# you use to collect yea st s$ &o ul# s>

??????????????????

(at ag ar oul# you use to collect !ta' (ylococci>

??????????????????

!a&'le "or 10 &inutes set ting t(e 'u&' a t 30l$&in%



29/60

alculate t(e /olu&e o" air sa&'le# in

& 3???????????????????????

@;'ress you resul ts a"ter incubat ion as s$& 3 air%

#'# #$ra%e * 'ir * sa+&l er..(is unit collec ts 'a rticl es car ryi ng &icroorgan is&s by

i&' action onto t(e sur"ac e o" ag ar &e #iu & in regular

contac t +A, 'lates %

.(e ag ar & e# iu & ca n be sel ec te # to reco /e r any gro u' o"

&icroorganis&s%

Bn t(is 'ract ical use .!A to reco/er bacteria

!abarou# e;trose agar to reco/er =east s$&oul#s

Mannitol !al t to eco/er Presu&'ti/e !ta'(ylococci %

!a&'le 1000 l o" air%

Bncuba t e t(e aga r 'la te s at t(e a''ro'ri at e te&'era t u res %

A"ter incubati on re'ort on t(e nu&ber o" & icroorgan is&s

reco/e re# 'er & 3 air%



30/60

Esti +a t i o n o bio b$ rd e n on &ro d$ % t s .Bn or#er to esti&a te t(e e;ten t o" conta &in ation on

'ro#ucts t(e articl e in *uestion &ust be rinse# in #iluent

an# t(e nu&bers o" s #e te r&ine# eit(er by 'our ' la te

&et(o# or &e&bra ne "iltra tion%

Met(o#%

Pre'ara t ion o" 'ro#uct "or biobur#en is usually car ri e# out

in t(e la&inar "lo sa"ety (oo#%

A sa&'le o" 'ro#uct is &ay be c(o''e# using a sterile scissors an# t(e

'ieces 'lace# in one litre o" #iluent%

sually three pieceso" 'ro#uct are assaye# an# t(e result is e;'resse#

as t(e &ean 'er one ite& o" 'ro#uct%

.(e #ilue nt is s(a -e n "or 15 &ins to #islo#g e att ac ( e#

&icroorganis&s%

A% Pass one ali*uot o" 250 &l o" #iluent t(roug( a

&e&br an e using a sterile &e &br a n e "iltration

a''aratus %

Plac e t( e &e &b r a n e car e"ully gri# si#e u' onto t( e

sur"ace o" a .!A 'late%

abel an# incubate aerobically 37 "or 48 (rs%

@;'ress your resul t as $lit re o" #iluent i%e% 'er a&ount

o" 'ro#uct in t(e #iluent or 'er in#i/i#ual 'ro#uct %

B. Pass a second aliquot o" 250 &l o" #iluent t(roug( a


a''aratus %Plac e t( e &e &b r a n e car e"ully gri# si#e u' onto t( e

sur"ace o" a .!A 'late%

ab el an # incu ba t e anaerob i ca l l y in a gas Car 37

"or 48$72 (rs%

C. Pass a third aliquot o" 250 &l o" #iluent t(roug( a


a''aratus %

Place t(e &e &b ra n e onto t(e sur"ac e o" a !abarou #



31/60

e;trose agar 'late %

abel an# incubate aerob i ca l l y 25 "or 5 #ays%

A"ter incub atio n coun t all colonie s a'' e ar in g on t( e

&e &b ra n es an# e;'re ss t(e biobur# en as s 'er'ro#uct%

it( t(e re&ai ni ng #iluen t car ry out a 'la te coun t using

t( e sa & e ag ar &e #i a%

Pre'are a 1:10 #ilution o" t(e #iluent using 9 &l ingers%

Bn tri'licat e a## 1 &l o" t (e original sa&'l e an# t(e 1:10

#ilution to t(ree agar 'lates %

are"ully a## &olten .!A sirl allo to set an# incubate 37D "or 48 (rs%


#ilution to t(ree agar 'lates %

are"ully a## &olten .!A sirl allo to set an# incubate

37D in t(e gas Car "or 48$42 (rs%


#ilution to t(ree agar 'lates %are"ully a## &ol ten !aborau# e;trose agar sirl a llo

to set an# incubate 25D "or 5 #ays %



32/60

Res$lts:'ir 'nalysi s :

Biotes

tCFU /m3

)il ter

CFU /m3

#' #

CFU /m3

Casell

aCFU /m3

'nders

onCFU /m3

'nderson

E non

res'irab

le

E

res'irab

le

Total bacteria

Yeasts/moulds

Mannitolfermenters

TotalMicroorganisms

#! abs:#ete r&ine t(e sabbe# ar ea %

C),- / %+ 0

#$r a% e

s ! a b b e d

%olior+ s 1otal ba%t eri a

Conta% t &l ate s .#ete r&ine t(e contact ar ea %

C),- %+ 0

#$r a% e%ont a% t ed

1otalba%ter ia

+ an ni t ol er+ent e

rs

%olior+s

yea s t s and+o$lds

Co++ en t on res $l ts :



33/60

Personal Hygiene%

C), &er handNa+e beor e !as h ater !as h


Bio b$ rd e n o n &r od $ % tC),-&rod$%t

ba%ter ia

des%ri& t ion o &rod$% t 'erobi% 'naero b i % yea s t s - + o $ld s




34/60

Mi%ro bi o l o g i % a l 'nal y si s o #oil s an d

# e d i + e n t s .

Bn t(ese 'rac ti cal s e ill ana ly se soils an# se# i&en t s "or a / ari ety

o" &icrob ial 'o'ula t ions in or#er to get so&e i#ea o" t(e #i/ersi ty o"&icr oor ga ni s &s 'r es e nt in t( es e e n/ir on & e n ts % Bt is 'os si bl e to

"urt( er a na lys e t( es e soils to #isco/ er t( e "unc tio ns t( at t( es e

&icro bial 'o 'u la t io n s a re r es' o ns ible "or in t( ei r n atu ral ( ab it a t

i%e % r ecyc ling carbo n ni trog en sul'( u r an# '( os' ( o rus as ell a s

'ro#uction o" organic aci#s an# gass es an# &obil i< ation o" &et als

an# &icrob ia l cor ro sion % eg ra# a t io n or # e to ;i "ica tion o" a i# e

/arie ty o" to;ic org anic c(e &i ca ls inclu#in g (y#r oc ar b on s F

ali' (a ti c ar o& a t ic a n# ( al og e n a t e # ar e als o car ri e# out by t( es e

'o 'ula tions%

!oils are a co& 'l e; an# (et er og e no u s en/iro n& e nt cont ai nin g

&a ny #iscontin uo us &icro( abit at s an# t(er e"or e 'res en ts a#i""icult c(al lange to t(e in/est igator %

Bn t(e "irst 'ract ical #eter&ine t(e "olloing:

Mi%robial Po&$la t i o n Metho d Medi$ +.otal )acterial Gu&bers

's yc (ro'(iles s'rea # 'late 'e'ton e ye as t

e;t rac t agar%

&eso' (iles Pour 'late 'e'ton e yeas t e;trac tagar%

t(er &o ' (iles Pour 'late 'e'ton e yeas t e;trac t

agar%

.otal ungi+=eas ts $Moul#s, Pour 'late Malt Agar+aci#i"ie#,

Actino&yc e t e s Pour 'late Actino&yc e t e agar

Method: ar e" ully eig ( out 10 g o" s oil a n# a ## to 90 &l #ilu en t in !id e

ne%(ed "las-es%

Mi; b y gen tl e s(a-in g "or 5 &ins% to #is' er se &icroor ga ni s &s

into sus' en s io n % Allo ( ea/ y ' ar ti cula t es to set tl e ou t% 1hi s is

th e 23 42 dil$tio n % Go car ry ou t ser ia l # ilu tion in 90 &l # iluen t s to 1 0 6 %

5 )or the ba%t eria using t(e pour pla t e tec h n i q u e a# # 1 &l o"

sa&' le in # u'li ca te "ro & 1" $6% 1" $5% 1" $4% 1" $3 to e ight ' e tr i #is( es

labelle# "or 22 re' ea t "or 32 an # 5 5 % A## coole# &ol ten'e'ton e ye as t e;trac t agar &i; care"ully%



35/60

(e n co &'l et e ly soli# in/er t a n# incu ba t e seri es at 22 32

a n# 55 e;a&ine reg ula rly u ntil n o "ur t( e r colo nies a'' ear on

t( e 'la te s a n# not e t (e nu &b e r s %

or t(e &sy%hro&hi le s t( e 'o ur 'la te t ec (ni *u e ca nn ot b e us e#

as t(e te &' e ra t ur e o" t(e ag ar oul# -ill t (e (e at sen siti/ ebacteria% Usin g th e spr e a d pla t e te ch ni q u e 'i'et te 0%1 &l o "

sus 'e n si on "ro& 1" $5% 1" $4% 1" $3 an# 1" $2 onto t(e prepo&red

chilled agar an# s'rea# e/en ly it( t(e glass s'rea# er % Allo to

#ry an# incub a te a t 4 7 "or s e6 e n da ys .

)or th e $ ng i &sin g th e po &r plat e t ec hn i' & e a## 1 &l o"

s a& 'l e "ro & e ac ( #ilution 1" $5% 1" $4% 1" $3 an# 1" $2 1" $1 to two

'etri #is(es% A## coo le# &ol ten Malt Agar +aci #i"ie# 'H 4%5,

&i; care"ul ly% (en co&'lete ly soli# label in/ert an# incubate

'airs at 22 until t(e ne;t class%

)or t h e a %t in o + y % e t e s using t(e po&r plate techni'& e a# # 1

&l o" s a& 'l e "ro & e ac ( #ilutio n 1" $5% 1" $4% 1" $3 an# 1" $2 1" $1 to

two 'etri #is(es% A## co ole# &ol ten Actino&yc e t e agar &i;

care"ul ly% (en co&'lete ly soli# label in/ert an# incubate 'ai rs

a t 2 2 until t(e ne;t class%

e'or t t(e nu &b er s o" Is ' er g dry wei g h t of soil.

eter&in e t(e dr y ! t . o th e s oi l u sing 10g o" et soil in &eta l

tr ays in t( e o/ en a t 1 04 "or 2 4 (r s% an# #ry to co ns tan t eig( t

using a #esiccator %

Mea su re t( e 'H o" t (e soil a n# recor #% Ma-e a slurr y o" t (e soil

+10 g, in al 2solution +20 &l 0%01M al2,%

Media

Jlycerol aesin agar: in 1000&l #eion ise# ater #issol/e t(e

"olloing: 10 g glycerolF 0%3 g caesinF 2%0 g KG3F 2%0 g GalF2%0g K 2HP 4F 0%05g Mg! 4%7H 2F 0%02 g a 3F 0%01 g

e! 4%7H2F 18 g agar an# 50 &g cyclo(e;i&i#e% A"ter

autocla/ing a#Cust to 'H 7%0 it( conc% Hl%

Malt e;tract agar + Aci#i"y to 'H 4%5 it( tar tar ic aci#,

Pe'to n e yeas t e;t rac t agar : in 10 00 &l # eioni se# a ter #is sol/e t(e

"olloing : 5 g 'e' tone 3 g yeas t e;t rac t an# 15 g agar % A"ter

au tocla/ ing but (en coo l a## 10 &l 1%0M al 2% A"ter

autocla/ing a#Cust to 'H 7%0 it( conc% Hl



36/60

Res$lts

C),- g dry ! e i ght soi l

Ba%teria

#oil ty&e 'syc(ro'(ile

s

&es o' (iles t(er &o '(ile

s

'%tino+y%

etes

Ye

ld s



37/60

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 substratesin 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 enzymeamylaseto

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 22C for 48 hrs.

Include uninoculated plates as controls.

Dr. Michael A. Broa ders De pt . Environ m e n t al Sc ience. Sli go 37

.!A agar it(

eit( er s ta rc(

!ource o"

colonies "ro&

1

2

37


38/60

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.



39/60

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 rapidmethod 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

490nm.

METHOD.

FDA is dissolved in acetone 2mg/ml and stored as a stock solution (at-20C).

Replicate samples of soil (10g) are placed in conical flasks with 20ml

sterile sodium phosphate buffer 60mM, (pH 7.6).

To each sample,0.1 ml FDA is added (10 g/ml final concentration).

The flasks are incubated on the shaker at 27C 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 490nm 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.



40/60

I#OL'1ION O) B'C1ERIOPH'GE.

PRELIMIN'RY ENRICHMEN1 O) 7PH'GE.

a seage or ri/er ater is centri"uge# an# t(e su'ernatant collecte#% .(e

bacteria are re&o/e# "ro& t(e su'ernatan t eit(er by +e+brane iltration or

by ina%ti6ation $sing %hloroor+ +si; or se/en #ro's o" c(loro"or& are a##e#

to 10 &l o" sa&'le% .(e tube is s(a-en to ensure t(at t(e ater is saturate#

it( t(e sol/ent% .(e c(loro"or& is alloe# to settle%

Bn ater sa&'les (ere t(e concentrations o" /iruses is lo it &ay be

necessary to concentrate t(e /iruses by a#sorbing t(e& onto a &aterial suc(

as (y#ro;ya'atite or alu&iniu& sul'(ate%

2. A## t(e "iltere# sa&'le containing t(e /irus to about 20 g o"

(y#ro;ya'atite in a 1 l conical "las- an# s(a-e ra'i#ly "or 5 &ins% ollect t(e

(y#ro;ya'atite in a )uc(ner "unnnel an# #iscar# t(e "iltrate% .(e /iruses on

t(e (y#ro;ya'atite can be elute# using a #ilute '(os'(ate bu""er +0%8M

Ga 2HP 422%6g$100&l +80&l, L 0%8M GaH 2P424%96g$200 &l +20&l,,%

0. Gaturally occurring coli'(ages can be concentrate# accor#ing to t(e"olloing 'roce#ure:

2 &l o" a 10E Al2+!4,3 solution is a##e# to 1 litre o" t(e sa&'le t(e 'H is

a#Custe# to 5%5 it( Hl an# t(e sa&'le le"t o/renig(t at 6% .(e Al+H, 3

"locs (ic( (a/e "or&e# toget(er it( t(e a#sorbe# '(ages are centri"uge# at

3000 g "or 5 &in%

.(e se#i&ent is t(en resus'en#e# in 10 &l 0%1 M citrate bu""er it( a 'H o"

4%7%

1 &l concentrate is &i;e# it( 0%5 &l o" an E.colisus'ension containing 108$ &l an# it( 5%0 &l so"t agar% .(e tube containing t(e &i;ture is

t(oroug(ly &i;e# an# 'oure# into a Petri #is( containing 20 &l try'tone

soya yeast e;tract agar% .(e 'lates are counte# a"ter 24 (ours incubation at

37%



41/60

)rain Heart Bn"usion brot( +#ouble strengt(, +50 &l ! in 200 &l conical

"las-s, is 're'are# an# sterilise#%

.o t(is 50 &l o" t(e ater sa&'le sus'ecte# o" containing bacterio'(age is

a##e#%

A"ter &i;ing ell 20 &l o" a young culture o"E.coli +68 (rs ol#, in 'e'tone

ater is a##e# an# incubate# on t(e s(a-er table "or 810 (rs or until a

#ecrease in turbi#ity is obser/e#%

Pre'are a control to obser/e t(e grot( o"E.coli un#er t(e sa&e con#itions%

.(e analysis is carrie# out in #u' licate%



42/60

I#OL'1ION 'N8 EN,MER'1ION O) 7PH'GE.

A"ter incubation t(e bacteria are re&o/e# "ro& a s&all /olu&e o" brot( by

centri"ugation an# &e&brane "iltration%

.(e nu&bers o" bacterio'(age in t(e enric(e# brot( are #eter&ine# by

're'ar ing a series o" #eci&al #ilutions in 1$4 str% ingerIs solution%

Alternativel(% i# onl( thepresence or absenceo# the )acteriophage is re'&ired to )e

demonstrated% then a more rapid proced&re can )e carried o&t.

*ive ml o# the enriched )roth are trans#erred to a test t&)e and placed in a +ater )ath

at 56,- #or 3"min. his is s#icient treatment to eliminate the )acteria +hile

/eeping the )acteriophages active. he presence o# the vir&ses can )e detected )(

#ollo+ing the proced&re )elo+.

.(e nu&bers o" I'(age in t(e #ilutions can enu&erate# eit(er by a Miles 9

Misra s$ra%e dro& tec(ni*ue to inoculate lan cultures o"E.coli or by a &o$r

&late te%hni$e.

an cultures o"E.coli can be 're'are# by s'rea#ing 0%1&l o" a 24 (r% cultureo"E.coli onto t(e sur"ace o" a ell #rie# agar 'late% Allo t(e 'lates to #ry%

.(e I'(age sus'ension is t(en #ro''e# "ro& calibrate# #ro''ers onto t(e

sur"ace o" t(e 'late an# incubate# o/ernig(t at 37%

A"ter incubation t(e 'resence o" I'(age is s(on by a clear area or se/eral

s&all clear areas -non as PL';,E# (ere t(e lan o" bacteria (as been

lyse# by t(e I'(age%



43/60

'lternati6e +ethod.

.(e &o$r &late te%hni$e is carrie# out by inoculating 3 &l .!A +-e't &olten

at 50, it( 1 &l or 0%5 &l o" an o/ernig(t brot( culture o" t(e (ost

bacteriu& &i; ell to #istribute t(e cells e/enly in t(e agar% .o t(is is a##e#

0%5 &l $0%2 &l o" t(e bacterio'(age #ilution again &i; ell%

Work quickly to prevent the agar from solidifying, preferably in a warm

environment.

Pour t(e &olten agar onto t(e sur"ace o" a +armed .!A 'late an# incubate at 37

o/ernig(t% lear areas or 'la*ues (ere t(e bacteriu& (as been lyse# by t(e

I'(age are counte#%



44/60

Enri% h+ e n t Isol at io n O 8e gr a d e r s O

Organi% Conta+ina nt s

Bn t(is series o" 'ractic als you are re*uire # to

isolate an# 're'are a 'ure culture o"

&icroor ga ni s& ca' abl e o" grot ( on one o" t(e

organic c(e&icals liste# belo%

.(e 'roc e# u re "or isolatio n is on e o" enric( & e n t

isolation in batc ( cultur e alt(o ug ( so& eti &e s

contin uo us cultur e tec (ni *u e s are us e# to isolat e

organis &s it( a lo a""inity to t(e organicconta&inant in *uestion%

.(e sourc e o" t(e org anis & s "or t(is 'ra ctic al is

acti/ at e # se ag e alt(o ug ( soil re 'r es e n t s a /as t

reser/oir "or all -in#s o" &icroorganis &s%

Proce#ure:

1% .o t(e 50 &l acti/ate# seag e in a 250 &lconical "las- a## t(e c(e&ical un#er

in/e stig a tio n as t(e sole car bo n sour ce +200

&l$l,%

2% Bncub at e on an orbital s(a- er at 25 "or se/en

#ays%

3% A"ter incuba ti on str ea- a sa& 'le onto .!A #orsingle colon( isolation an# incubat e "or 4 7

#ays at 22 25% @;a&ine #aily "or grot(%

4% Many colonies &ay #e/elo' on t(e agar

&e#iu& an# all &ay be trea te# as sus'ect

colonies ca' abl e o" #egr a#in g t(e c(e &ic al

un#er in/est iga t ion as t(ei r sole carbon source %

5% !elect a single colon( "or in/est iga t ion #escribe



45/60

t( e colony &or '( olo gy an # tra ns" er a 'ortio n

into &ineral salts &e#iu& containing t(e

c(e&ical un#er in/estiga tion as t(e sol e

car bo n sour ce .

6% Bncu ba t e on t(e s( a- er tabl e e; a&i ne #aily an #

note turbi#i ty as e/i#ence o" grot( %

7% !trea- aga in on to .!A #or single colon( isolation

an# incub at e "or 4 7 #ay s at 22 25% Again

e;a&ine #aily "or grot(%

8% e' ea t ste 's 56 an# 7 until you are con"i#e ntt(at t(e cultur e gros in t(e &in er al &e #iu &

using t(e selecte # c(e&ical as sole carb o n

source %

9% @;a&in e t(e cell &or '( olo gy an# re'or t Jra &

stain s(a'e an# si


46/60

(ec- t(e M!! s(eets "or sa"e ty #ata to;ici ty le/els an# any

ot(er rel e/ an t # ata %



47/60

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 thenitrogen 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 ofBacillus cereus, Pseudomonas

fluorescens andProteus vulgaris, spotting tile, Nesslers reagent and pH

paper.

Procedure. Inoculate tubes of peptone with each of the above and

incubate at 27C. Remember to leave one tube as an uninoculated

control. Test for the presence of NH3with Nesslers reagent on a spotting

tile.

Note the pH, record the results.



48/60

2. NITRIFICATION.The transformaton of NH3to N0-3in soils is carried out by highly

specialized aerobic, autotrophic bacteria.Nitrosomonas derives itsenergy for growth by the oxidation of NH3to N0-2, andNitrobacter

completes theoxidation of N0-2to 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 NH3to N0-3by

using enrichment shaker flask culture.

MATERIALS. 2 x 250ml conical flasks containing 100 ml of the

enrichment medium as detailed below.

NITRITE FORMATION MEDIUM NITRATE FORMATION

MEDIUM

(NH4)2SO4.................. .1.0g

NaNO2............................0.5gK2HPO4..........................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,



49/60

Sulphanilic acid, naphthalamine, diphenylamine in conc. H2S04, urea

powder, dil. H2S04and test tubes.

PROCEDURE.

To each flask add 9.0 g of test soil. Incubate the flasks on the shakertable at 220 r.p.m. and at 27C.

Each flask should be tested for the presence of NH3, N0-2and N0-3using

the spotting tiles and the procedure as detailed below.

Present the results in a table, showing the appearance of N0 2and N03during the test period.



50/60

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 andthe culture of Pseudomonas. and incubate at 27C. Remember to retain

one tube as an uninoculated control. Test for the presence of nitrate and

nitrite during the incubation period. Tabulate your results.



51/60

Qualatative tests for ammonia. nitrite and nitrate.

Test for Ammonia.

Put a drop of culture solution onto a spotting tile. Add one drop ofNessler'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 bepresent by boiling a little of the culture solution with urea plus dil.H2S04in a test tube. Cool and repeat the test for Nitrate.



52/60

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 isa highlyaerobic, 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 ofAzotobacter from soil.

Mannitol............................................................. ..10 g

or

Sucrose............................................................... ..1 0 g

K2HP04.............................................................. ...0.5 g

CaC03................................................................ ...0.5g

MgS04.7.H2O..................................................... ...0.025g

FeS047.H2O........................................................ ...0.025gNa2MoO4........................................................... ....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 27C until the next practical

period and look for typicalAzotobacter colonies. These should appear as

raised, moist, glistening white colonies. Examine the cell morphology

by Gram staining.



53/60

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 andAzotobacter

isolated and purified by repeated subculture in shake flask culture.



54/60

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 CFUper 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.



55/60

Medium for the isolation of Actinomycetes.

DEXTROSE NITRATE ACTIDIONE AGAR.

Agar---------------------- l5 gDextrose------------------l gKH2PO4----------------------0.1 g

NaN03--------------------0.1 g

KCl -----------------------O.l gMgS04.7H20-------------O.l g

H2O-----------------------l l

pH-------------------------7.0

Autoclave 15 lbs / 15 mins.

Acid PDA.

To Potato Dextrose Agar add sufficient sterile H2S04to 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 10 g

NH4NO3 1.0 g

Yeast extract 2.0 g

MgSO4.7H2O 0.2 g

KH2PO4 3.0 g

FeSO4.7H2O 0.0025 g

Water 1 literAgar 15 g

Rose Bengal Dye 0.09 g

Neomycin 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 90ml of diluent and shake vigerously

for 10 mins to suspend the soil completely. Allow the large particulates



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to settle and continue decimal dilution to 10-7.

Prepare pour plates in duplicate using 1 ml samples of appropiate

dilutions into the selected agar media. Incubate at 25C and examine the

plates periodically for development of the colonies. Tabulate your

results.



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@nric( &e nt Bsolation o" #egra # es o" organics

suitable "or )iore&e #ia tion%

Bn t(is series o" 'ractic als you are re*uire # to

isolate an# 're'are a 'ure culture o"

&icroor ga ni s& ca' abl e o" grot ( on one o" t(e

organic c(e&icals liste# belo%

.(e 'roc e# u re "or isolatio n is on e o" enric( & e n t

isolation in batc ( cultur e alt(o ug ( so& eti &e s

contin uo us cultur e tec (ni *u e s are us e# to isolat e

organis &s it( a lo a""inity to t(e organic

conta&inant in *uestion%

.(e sourc e o" t(e org anis & s "or t(is 'ra ctic al is

acti/ at e # se ag e alt(o ug ( soil re 'r es e n t s a /as t

reser/oir "or all -in#s o" &icroorganis &s%

Proce#ure:

1% .o t(e 50 &l acti/ate# seag e in a 250 &l

conical "las- a## t(e c(e&ical un#er

in/e stig a tio n as t(e sole car bo n sour ce +200

&l$l,%

2% Bncub at e on an orbital s(a- er at 25 "or se/en

#ays%

3% A"ter incuba ti on str ea- a sa& 'le onto .!A #orsingle colon( isolation an# incubat e "or 4 7

#ays at 22 25% @;a&ine #aily "or grot(%

4% Many colonies &ay #e/elo' on t(e agar

&e#iu& an# all &ay be trea te# as sus'ect

colonies ca' abl e o" #egr a#in g t(e c(e &ic al

un#er in/est iga t ion as t(ei r sole carbon source %

5% !elect a single colon( "or in/est iga t ion #escribe



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t( e colony &or '( olo gy an # tra ns" er a 'ortio n

into &ineral salts &e#iu& containing t(e

c(e&ical un#er in/estiga tion as t(e sol e

car bo n sour ce .

6% Bncu ba t e on t(e s( a- er tabl e e; a&i ne #aily an #

note turbi#i ty as e/i#ence o" grot( %

7% !trea- aga in on to .!A #or single colon( isolation

an# incub at e "or 4 7 #ay s at 22 25% Again

e;a&ine #aily "or grot(%

8% e' ea t ste 's 56 an# 7 until you are con"i#e ntt(at t(e cultur e gros in t(e &in er al &e #iu &

using t(e selecte # c(e&ical as sole carb o n

source %

9% @;a&in e t(e cell &or '( olo gy an# re'or t Jra &

stain s(a'e an# si


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(ec- t(e M!! s(eets "or sa"e ty #ata to;ici ty le/els an# any

ot(er rel e/ an t # ata %



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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 filterpaper/ caesin.

Put the mixture into a 500 ml measuring cylinder and fill with fresh

water.

Expose the column to natural daylight at room temperature.

Sem 2 Practical

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