Microbiological Medium

Background on media:

Sincebacteriawerefirstdiscoveredand studied,therehasbeenapushtodevelop ways to cultivate theorganisms in thelaboratory.Microorganisms, likeallorganisms, have requirements for theirgrowthand maintenance.Itwasthereforethechoreoftheearlymicrobiologists todiscoverwhatthoserequirements were and to developsuitable media to allow for the growth ofthevariousbacteriaandothermicroorganisms the wanted to cultivate.

Overtheyearshundredsofdifferentformulas for media have been developedtocultivateall manner ofbacteria,fungi,algae,etc… Some ofthese media aresimple in nature made up of knownamounts ofsalts,sugarsandotherorganic molecules like vitamins.Thesesimple or defined media are fine forgrowing manyorganisms, but there aresome bacteria in particular that havemore demandingrequirements. Forthese organisms, more complexmediawere developed using infusions ofanimal andplantpartsandoftensupplemented with blood or serum.Thelist of ingredientsforsome ofthesemediamay sound a bit like a witch’sbrew; brains of calves, the blood of sheep, and so forth...

Another important concept when dealingwith mediadevelopment is to determineif the medium needs to be a liquid(broth)orasolid(agarbased).Brothmedia are most commonly used tocultivate purecultures inthe laboratoryor to investigate a variety of biochemicalreactions.Agar based media are oftenused to isolate organisms froma mixed

culture or contaminatedsample.

Besidesthegeneral make upofthemedia, thereare other considerations thatone must lookat.Agentssuchassaltorantibioticscanbeaddedtoa medium togive it a selective property.Selectivemedia allow for the growth of someorganisms, butinhibitthegrowthofothers.Thesetypesof media areveryusefulwhentryingto isolate a bacteriumfroma mixed culture or contaminatedsample.

Anotherconsiderationistheuseofdifferentiation agentsinthe medium todetermine different patterns of growth ormetabolism. Differential mediumusualhave some formof indicator in themthatallows foreasydetermination ofthemetabolic activityoftheorganismgrowing on it. These mediumare usefulin identifying unknown organisms.

Sometimesselectiveagents can be addedto a differential mediumto make is bothselective and differential.

Inthis laboratory we willbelookingatbacteriologicalmedia and examiningexamples of and uses for general labmedia, selective media, and differentialmedia.

The firstplace to start is with the actualpreparationofthe media.Theabsolutefirst stepistoREADTHEINSTRUCTIONSprinted on the bottleof medium. The instructions will outlineexactlywhatyouneedtodoinorderto properly make up the medium. There isusuallyalistofingredients,stepstofollow in the preparation, how themediumis to be sterilized, and how and

when any additivesare to be added.Belowisan example ofthelabelfromabottle of media.

Figure 1– Label froma bottle of EMBagar

Materials:

1)1 plate plainagar

2)1plateM-9agarwithglucose

3)1 plate nutrient agar

4)1 plate blood agar

5)1plateMSA

6)1plateEMBagar

7)1 plate DNase agar

8)2 tubes of TSI agar slants

9)2 tubes of BHI broth

10)2 tubes of urea broth

11)bacterial cultures:

a)S. aureus

b)S. epidermidis

c)E. coli

d)P. vulgaris

e)S. pyogenes

f)P. aeruginosa

g)S. typhimurium

h)Mixed culture

12)Gramstain reagents

13)glass microscope slides

14)wax pencils

15)Bunsen burner

16)inoculatingloop

17)Kovac’s reagent

Day One Protocol:

1)obtainall the necessary media andlabeleachplateandtubewithyour initials

Nutritional Requirements of BacteriaIn this section we will beinoculatingseveral different media with 4 differentbacteria and determining iftheywillgrowonthedifferent media.Eachmedia has a different level of nutrientsandeach organismhasadifferentsetofnutritional requirements.To set up thissectionofthelabfollowthese steps:

1)usingyourwaxpencildividethe followingplatesinto4equalportions and label each quadrant withtheinitials of one of the organisms to beused – Pa, Spy, Se or Ec

a)plain agar

b)M-9 plus glucose agar

c)nutrient agar

d)blood agar

Be sure to write on the bottom of theplates not the tops

2)usingaseptictechnique,inoculate each plate inthe appropriatequadrant with a small amount of thebacterialculture.The instructor willgive specific instructions on how toperformtheinoculation.Be sure not tospreadtheorganismbeyondthebounds of the quadrant.

3)incubatetheplatesat 37oC overnight

IsolationofBacteriafromMixed Cultures

In this section we will beusing twoselectivemedia to isolatetwo differentbacteria from a mixed culture.

1)inoculateaMSAandanEMBplate with a samplefromtheMixedculture.The instructor will givespecificinstructionsonhowto performthe inoculation.

2)performa Gramstain on the mixedcultureandrecordyourresults below:

3)incubatetheplatesat 37oC overnight

Theuseofdifferentialmedia

Inthis section wewillbelookingatthedifferences in growth and reactions ofseveral different bacteria on a number ofdifferentmedia.We will be lookingfordifferences in carbohydratefermentation, productionofbiologicalend products, and the production ofenzymes.

Triple Sugar Iron Agar TSI media contains three sugars(glucose,lactoseandsucrose)andan ironingredient for detecting H2S

production - thus the name Triple SugarIron.Ifthesugaris fermented themediumwill turn fromorange-red ortoyellow.If H2S is produced the mediumwill be blackened.If the sugars are notfermented, the media will turn a pink orred color.

1)ObtaintwoTSIagar slants andinoculate.Itis important thattheinoculationbedoneexactlyas described below:

2)aseptically take a loopful of bacteria fromtheE. coli(Ec) culture andgently stab it into the butt ofthe

will givemore details onhow toproperlyinoculatethesetubes.

3)repeattheprocessusingthe

Salmonella(St) culture.

4)incubatethetubesat 37oC overnight

Figure 2–TSItube.Notethecolorof the mediumin the area of the slant andthebutt.Besuretorecordand blackeningofthe medium asthisindicatestheproductionofhydrogensulfide gas.

Indole Production

Someorganisms will converttryptophanintoindole.Inordertoseethis,theorganisms need to be grown in amediumthat is high in tryptophan suchas BHI broth. To determine the presenceof indole after incubating the organism overnight, several drops of Kovac’s reagent is added tothetubeand observed for a red colored ring to formontopofthemedium.

1)Obtain two tubes of BHIbroth and inoculatewithacoupleofloopfulsof the E. coli(Eca) andSalmonella(St)culture.

slant.Remove the loop and gentlyinoculate theslant. The instructor

2)incubatethetubesat 37oC overnight

DetectionofBacterial EnzymesWe will be using two media to detect thepresence of the enzymes DNase andurease. Both of these enzymes are usedintheidentificationofavarietyofclinically significant bacteria.

DNase Production

The detection of the enzyme DNase,which degrades polymerized DNA), canbe done in a variety of ways. We will beusing a special mediumthat containsDNA and methyl green.Methylgreen onlybindstohighly polymerized DNAand the color will fade if the DNA isdegraded.

1)obtain one plate of the DNasemediumanddivideitinhalfwithyour wax pencil.

2)usingaseptictechnique,inoculatethe plateintheappropriatehalfwithasmallamount ofthe bacterialculture

S. aureus (Sa) and S. epidermidis(Se).Theinstructorwillgive specificinstructionsonhowto performtheinoculation.Be sure not tospreadtheorganismbeyondthe bounds of the half.

3)incubatetheplatesat 37oC overnight

Urease Assay

Ureaseisanenzyme thatbreaks downurea into ammonia and carbon dioxide. As the ammonia content of the mediumincreases, sowill the pH ofthe medium.A pH indicator will detect this change andindicatethat the urea is being degraded.

1)Obtain two tubes of urea broth and inoculatewithacoupleofloopfulsof the E. coli(Ec) andP. vulgaris(Pv)culture.

2)incubatethetubesat 37oC overnight

NOTES

Day Two:

You will need to examine your platesand tubes and record your results inthetables below.

Nutritional Requirements of Bacteria

1)examine yourplatesforgrowth.Growth is indicated by colonies on the medium.

2)record your resultsin table 1.IsolationofBacteriafromMixed Cultures

1)observe your plates for growth and recordwhatthecolonieslooklike.

2)performa Gramstain onrepresentative colonies fromeachplateandrecordyourresultsintable 2.

TheuseofdifferentialmediaTSI agar

1) observeyourtubesandrecordyour resultsin table 3.

Indole production

1)observe your tubes for growth and addabout5dropsoftheKovac’sreagent

2)gentlyshakethetubeandobservefor the presence of a red color at the top

ofthetube.Aredcolorindicatesa the presence of indole.

3)record your resultsin table 3.DetectionofBacterial EnzymesDNase agar

1)observetheplateforgrowthanddeterminethe color of themediumadjacent tothecolonies.Agreencolor indicates noDNase activityand a clearing of the mediumindicatesthepresenceofDNase.

2)recordyourresultsinTable 4.

Urease broth

1)observe your tubes for growth and color change. If the mediumlooksthe same aswhenitwasinoculated,salmon color,itisnegativeforureaseactivity.Ifthemediumhasa bright pinkcolor, it is positiveforurease.

2)recordyourresultsinTable4.

Table 1 - Nutritional Requirements of Bacteria

Medium / Organism
Pa / Spy / Se / Ec
Plain agar / Growth [ ]
No Growth [] / Growth [ ]
No Growth [] / Growth [ ]
No Growth [] / Growth [ ]
No Growth []
M9 + glucose / Growth [ ]
No Growth [] / Growth [ ]
No Growth [] / Growth [ ]
No Growth [] / Growth [ ]
No Growth []
Nutrient agar / Growth [ ]
No Growth [] / Growth [ ]
No Growth [] / Growth [ ]
No Growth [] / Growth [ ]
No Growth []
Blood agar / Growth [ ]
No Growth [] / Growth [ ]
No Growth [] / Growth [ ]
No Growth [] / Growth [ ]
No Growth []

Table 2 - Isolation of Bacteria from Mixed Cultures

Medium / MSA / EMB
Gram Stain Results

Table 3 - The use of differential media

TSI agar / Organism
Ec / St
Butt / Yellow[] Red [ ]
H2S [ ] / Yellow[] Red [ ]
H2S [ ]
Slant / Yellow[] Red [ ]
H2S [ ] / Yellow[] Red [ ]
H2S [ ]
Indole Test / Ec / St
Indole Production / Yes [ ]
No [ ] / Yes [ ]
No [ ]

Table4-Detectionof BacterialEnzymes

Test / Organism
Dnase / Sa / Se
Presence of Dnase / Present[] Absent [ ] / Present[] Absent [ ]
Urease / Ec / Pv
Presence of Urease / Present[] Absent [ ] / Present[] Absent [ ]

Questions

1)Didallthe organisms testedgrow equally wellonallthe media used?

2)Wasthereadifference between the organisms growing on the EMB and MSA plates?

3)Wasthereanoticeable difference betweentheEcandStontheTSIagarslantsandinthe indoletest?

4)Could easilydifferentiate between Sa and Se based on their reactions on the DNasetestagar?

5)Was there a difference between Ec and Pv when grown in the urease medium?