micro lab packet updated january 2016

7/24/2019 Micro Lab Packet Updated January 2016

http://slidepdf.com/reader/full/micro-lab-packet-updated-january-2016 1/31

Microbiology Lab Packet

Students are responsible for all the material in this lab packet.

Last reviewed: January 2!"



M#$ %2 Laboratory &ules1. Backpacks, purses, and other personal efects will be placed on the rack near

lab entrance when you rst come in to lab. Paper and writing utensils are

provided at the bench.2. No ood !including gum" or drink allowed in the lab.#. $lose toed shoes must be worn at all times%. &ong pants !to ankles" and sleeves !mid'arm or longer" must be worn to lab.(. )oggles will be provided in your bins, and must be worn at all times.*. )loves are provided and should be worn at all times.+. &ab coats are provided and should be worn at all times. &ab coats are N- to

be removed rom lab.. $ell phone use is not allowed in lab at any time. -hey should be on silent and

put away. /n the event an emergency should arise and you need to use your

cell phone, let your -0 know, remove your gloves and lab coat, wash your

hands, and leave the lab.. -here will be no talking during the lecture portion o lab sessions. Paying

close attention during the lectures will improve your total grade or the

course.1.&ong hair should be tied back during lab.11.-est tubes should never be carried without a test tube rack. 3o not pick up

test tubes by the cap.12./n the event o a spill do not move. Notiy the -0 and people around you

immediately.1#.$ough drops, eye drops, and other orms o oral medication may not be

consumed during lab.1%.4ash hands beore and ater each lab.



Simple Microscope: one lens#ompound Microscope: two lenses

1. Ocular-eyepiece lens (usually 10x)2. Objective-nosepiece lenses (commonly 4x 10x 45x 100x (oil immersion))

o Total a!ni"ication # Objective a!ni"ication x Ocular a!ni"ication

+mportant Parts of the Microscope:

ramework inclu$es t%e arm an$ base

o &tructural parts o" t%e microscope w%ic% support t%e basic "rame.

Stage %ol$s t%e sli$e

o T%e mec%anical sta!e clamps t%e sli$e an$ moves t%e sli$e aroun$ t%e sta!e.

Lens system inclu$es

o ,culars ' eyepiece lenses (usually 10x ma!ni"ication)

o ,b/ectives ' lenses attac%e$ to rotatable nosepiece common ma!ni"ications o" 4x 10x 45x (low

power %i!%-$ry objectives) an$ 100x (oil immersion lens).

ar"ocal microscope "ocusin! a$justments are not to be ma$e w%en c%an!in! objective

lenses.

Oil immersion lens uses oil wit% approximately t%e same re"ractive in$ex as !lass to

prevent li!%t loss $ue to $i""raction (ben$in! o" li!%t rays) w%ic% woul$ occur i" li!%t

travele$ "rom one re"ractive in$ex to anot%er (e.!. !lass to air).

*s ma!ni"ication o" t%e objective lens increases t%e wor+in! $istance ($istance between

t%e object on sli$e an$ t%e objective lens w%en in "ocus) $ecreases.

o #ondenser ' $irects li!%t towar$s t%e objective lens in bri!%t "iel$ microscopy

,n $ar+ "iel$ microscopy t%e con$enser $irects li!%t at obliue an!les away "rom t%e

objective lens in a manner t%at allows only objects in t%e "iel$ o" view to re$irect or

scatter li!%t into t%e objective lens. T%is causes objects to appear w%ite on a $ar+ "iel$. T%e iris diaphragm (lever locate$ in t%e con$enser) a$justs t%e $iameter o" t%e cone o"

li!%t so t%at it just "ills t%e objective lens.

• *s you close $own t%e $iap%ra!m

1. T%e li!%t intensity $ecreases

2. /ontrast improves

. ept% o" "iel$ increases4. imit resolution (wit% oil immersion lens)



$-#(&+-L M,&P3,L,45

i!%t microscopy reveals t%ree principle "orms o" microor!anism

o ore or less sp%erical or!anisms #,##+

o /ylin$rical or!anisms $-#+LL+

o &piral s%ape$ 3(L+#,+*-L

,ncompletely separate$ cocci may appear in a number o" $i""erent patterns $epen$in! upon t%e plane in

w%ic% t%ey $ivi$e an$ %ow t%ey remain attac%e$

o *iplococci (pairs) ' $ivi$e in one plane

o Streptococci (c%ains) ' $ivi$e in one plane

o etracocci (tetra$s) ' $ivi$e in two planeso Staphylococci (clusters) ' $ivi$e in t%ree planes irre!ularly

o Sarcinae (cuboi$al pac+ets) ' $ivi$e in t%ree planes re!ularly

7acilli can appear in a number o" $i""erent cylin$rical s%apes

o #occobacilli are very s%ort an$ almost appears sp%erical but t%ey are just sli!%tly lon!er in one

$irection t%en t%e ot%er.

o usiform bacilli are tapere$ at t%e en$s appearin! as "ootball li+e in s%ape.

o ilamentous bacillary forms !row in lon! t%rea$s.



S-+6+64 P&,#(*7&(S

ost microor!anisms are $i""icult to see usin! li!%t microscopy $ue to t%eir si8e an$ t%e lac+ o" contrast between t%e cell an$ t%e environment. T%e contrast is improve$ wit% t%e %elp o" $yes. yes are or!anic compoun$scontainin! a c%romop%ore wit% a""inity "or cellular material.

ypes of *yes:

#ationic (basic $yes positively c%ar!e$ c%romop%ore): met%ylene blue crystal violet

-nionic (aci$ic $yes ne!atively c%ar!e$ c%romop%ore): aci$ "uc%sin /on!o re$ 6i!rosin

at Soluble (no c%ar!e): &u$an blac+ stains !ranules o" oly-9-O:-butyric aci$

+nsoluble *yes (water insoluble): ,n$ia in+ (colloi$ suspension o" carbon particles)

ypes of Staining Procedure:

!. 6egative Staining

&tains t%e bac+!roun$ an$ not t%e cell in bri!%t-"iel$ microscopy (not t%e same t%in! as $ar+ "iel$

microscopy). oes not reuire %eat "ixation t%ere"ore $oes not $istort si8e or s%ape o" cells.

Two $yes use$

o 6igrosin ' a blac+ anionic (ne!atively) c%ar!e$ $ye. T%e ne!atively c%ar!e$ $ye is repelle$ by t%e

ne!atively c%ar!e$ sur"ace o" t%e bacterial cell.

o

+ndia ink ' an insoluble $ye (a colloi$al suspension o" carbon particles) w%ic% $oes not penetrate t%ecell sur"ace.2. Simple Staining

One $ye use$ to stain all cells t%e same color. /an be use$ to tell morp%olo!y (s%ape) an$ si8e ;alt%ou!%

ne!ative stainin! is better "or si8e<. /ationic $yes are positively c%ar!e$ an$ are attracte$ by ionic "orces to

t%e ne!atively c%ar!e$ sur"ace o" t%e bacterial cell.

Two commonly use$ $yes are met%ylene blue an$ crystal violet.

%. *ifferential Staining

&tainin! proce$ure w%ic% causes cells to stain $i""erently base$ on properties o" t%e cell.

Two examples o" $i""erential stainin!

o -cid ast Stain ' $i""erential stain proce$ure t%at causes cells to stain $i""erently base$ on

c%aracteristics o" t%eir cell wall. *ci$ "ast microor!anisms %ave a %i!% wax content in t%eir walls

w%ic% reuires t%e use o" steam to allow $ye to penetrate t%e cell. /ells are steame$ in t%e presence o"

carbol "uc%sin an$ $ecolori8e$ wit% aci$ alco%ol /ells w%ic% are aci$ "ast (microor!anisms %ave a



o 4ram Stain ' $i""erential stain proce$ure t%at causes cells to stain $i""erently base$ on c%aracteristics

o" t%eir cell wall. =ram-positive microor!anisms %ave a %i!%er pepti$o!lycan an$ lower lipi$ content

t%an =ram-ne!ative microor!anisms. /ells are staine$ wit% crystal violet t%en "ixe$ wit% io$ine

"ormin! a crystal violet-io$ine complex wit%in t%e cell. 3t%anol is t%en a$$e$ as a $ecolori8er. =ram-ne!ative cells are easily $ecolori8e$ because t%e et%anol $issolves t%e %i!% lipi$ cell wall allowin! t%e

crystal violet-io$ine complex to rea$ily exit t%e cell. T%ese cells are t%en counterstaine$ wit% &a"ranin.

=ram-positive cells resist $ecolori8ation $ue to t%e $i""erence in cell wall consistency retainin! t%e

crystal violet-io$ine complex.

Stain 4ram 081 4ram 091

Primary stain /rystal violet purple purple

Mordant ,o$ine purple purple*ecolorier 3t%anol purple colorless

#ounterstain &a"ranin purple pin+

;. Structural Staining

Spore Staining < some microor!anisms pro$uce %eat an$ c%emical resistant structure$ calle$

en$ospores or "ree spores. To stain t%e spores t%e cells must be steame$ to allow "or t%e $ye (malac%ite!reen) to enter t%e spores. Once t%e spores are staine$ all ot%er microor!anisms an$ ve!etative cells can

easily be $ecolori8e$ wit% water w%ile t%e "ree spores an$ en$ospores retain t%e malac%ite !reen. T%e

ot%er microor!anisms an$ ve!etative cells are t%en counterstaine$ wit% &a"ranin.

o (ndospores appear as a !reen center wit%in a pin+ sporan!ium

o ree spores appear as small !reen oval bo$ies

o T%ree !enera o" spore "ormin! or!anisms

Bacillus ' aerobic =ram-positive ro$

• *erobic !reen # en$ospore"ree spores o" Bacillus

• *erobic pin+ # ve!etativesporan!ia o" Bacillus

Clostridium ' anaerobic =ram-positive ro$

• *naerobic !reen # en$ospore"ree spores Clostridium• *naerobic pin+ # ve!etativesporan!ia o" Clostridium

Sporosarcina ' cocci



M(*+- -6* $+,#3(M+#-L (SS

T%ere are "ive met%o$s o" tube me$ia preparation1. Pour: 15 ' 20 m o" liui$ a!ar use$ to pour into a plate2. $roth: 5 ' ? m o" liui$ me$ia

. *eep: 5 ' ? m o" me$ia w%ic% %as soli$i"ie$ in an upri!%t position

4. Slant: 5 ' ? m o" me$ia w%ic% %as soli$i"ie$ at an an!le$ position

5. ermentation $roth: brot% wit% ur%am tube a$$e$

6atural media is compose$ o" complex raw materials w%ose actual c%emical composition is unknown.

• 3xample 6utrient *!ar (6*)

Synthetic media@s exact c%emical composition is known an$ in many instances is $esi!ne$ "or isolation selection or

$i""erentiation o" speci"ic types o" microor!anisms. Two main cate!ories area1 Selective media ' "avors t%e !rowt% o" one type o" microor!anism over anot%er. T%is is accomplis%e$ by

eit%er in%ibitin! unwante$ microor!anisms or enric%in! by provi$in! con$itions w%ic% are pre"erential to

t%e $esire$ microor!anism.b1 *ifferential media ' $i""erentiates or $istin!uis%es between $i""erent types o" microor!anisms base$ on

$i""erences in appearance o" !rowt% or color c%an!es.

Selective and>or *ifferential Media:

!. Phenylethyl -lcohol -gar 0P(-1

o &elects "or t%e !rowt% o" =ram-positive microor!anisms because p%enylet%yl alco%ol is in%ibitory to

t%e !rowt% o" =ram-ne!ative or!anisms.2. *(So?ycholate -gar 0*(S1

o &elects "or =ram-ne!ative microor!anism because $esoxyc%olate is in%ibitory towar$s t%e !rowt% o"

=ram-positive or!anisms.

o i""erentiates lactose-positivene!ative microor!anisms

actose "ermenters pro$uce aci$ w%ic% precipitates t%e bile salts in t%e me$ia an$ absorbs t%e

neutral re$ $ye t%ere"ore appearin! re$. 6on-"ermenters $o not $o t%is an$ $o not appear re$.

%. (osin Methylene $lue -gar 0(M$1

o &elects "or =ram-ne!ative or!anisms.



actose "ermenters pro$uce aci$ w%ic% precipitates t%e bile salts in t%e me$ia an$ absorbs t%e

neutral re$ $ye t%ere"ore appearin! re$pin+. 6on-"ermenters $o not $o t%is an$ $o not appear re$pin+ but will "orm colorless colonies.

=. $lood -gar

o i""erentiates microor!anisms base$ on t%eir reactions on bloo$. &%ows presence or absence o" an

exoen8ymes +nown as %emolysins t%at can brea+ $own %emo!lobin in bloo$.

4amma 0@1 hemolysis: no bloo$ %emolysis no 8one o" clearin! aroun$ t%e colony.

$eta 0A1 hemolysis: complete bloo$ %emolysis an$ complete clearin! aroun$ t%e colony.

:emo!lobin is completely $e!ra$e$.

-lpha 0B1 hemolysis: partial bloo$ %emolysis an$ partial clearin! aroun$ colony. artial

clearin! sometimes appears !reen $ue to partial re$uction o" %emo!lobin in bloo$. =reenis%

color is $ue to a partial brea+$own pro$uct o" %emo!lobin calle$ biliver$in.

$iochemical ests:

Tests use$ to $etermine p%ysiolo!ical c%aracteristics o" microor!anism particularly in terms o" bacterial en8ymesan$ t%e c%emistry o" bio-oxi$ation. 7ioc%emical tests on a!ar plates o"ten (but not always) loo+ "or t%e presence o"

exoen8ymes w%ic% are en8ymes secrete$ by t%e bacteria into t%eir environment to brea+ $own macromolecules

suc% as proteins an$ carbo%y$rates into monomeric subunits small enou!% to be transporte$ into t%e bacterial cell.

3n$oen8ymes are en8ymes active insi$e t%e bacterial cell to metaboli8e substrates insi$e t%e bacterial cell an$ can be $etecte$ in bot% soli$ me$ia an$ brot%s.

!. Starch -gar

o Tests "or t%e presence o" t%e exoen8yme amylase w%ic% %y$roly8es starc% to simple su!ars.

T%ese simple su!ars can t%en be transporte$ insi$e t%e cell.

o ,o$ine is a$$e$ to t%e starc% plate an$ appears blueblac+ w%en io$ine "orms a complex wit% starc%.

o ," amylase is present starc% will be %y$roly8e$ an$ t%e blueblac+ to purple color will not be observe$

aroun$ t%e colonies.2. Milk -gar

oTests "or t%e presence o" t%e exoen8yme caseinase w%ic% %y$roly8es casein (a pre$ominant protein in

mil+) into amino aci$ pro$ucts.o /asein !ives mil+ its w%ite color so a brea+$own in casein causes t%e mil+ plate to lose its w%ite color

an$ become clear aroun$ t%e caseinase positive colonies.%. Lipase Plate



De$ # ne!ative

/erise # al+aline

=. Methyl &ed 0M&1

o :/OO: E /O2 F :2

o Tests "or a mixe$ aci$ "ermenter w%ic% pro$uce $rastic amounts o" aci$ "rom t%e "ermentation o"

su!ars.

o T%is aci$ ultimately results in t%e lowin! o" t%e p: below 5.1 so w%en t%e in$icator met%yl re$ is

a$$e$ to t%e culture t%e met%yl re$ remains re$.

De$ is t%e only true in$icator o" a positive result.

o Escherichia is D positive.

". Doges9Proskauer 0DP1

o :/OO: E acetyl met%yl carbinol (*/)acetoin E 2-butane$iol

o Tests "or 2-butane$iol "ermenters w%ic% pro$uce less aci$ an$ more neutral pro$ucts t%an mixe$ aci$

"ermenters.

o 7ecause */ is easier to $etect t%an 2-butane$iol */ is teste$ "or w%en $eterminin! i" a

microor!anism is a 2-butane$iol pro$ucer.

o 7arritt@s rea!ents also +nown as G, (5-nap%t%ol) an$ G,, (HO:) are a$$e$ to t%e test culture.

o >%en oxy!en is present HO: (G ,,) will react wit% */ oxi$i8in! it to $iacetyl w%ic% reacts wit%

a !uani$ine-containin! compoun$ in t%e me$ia to pro$uce a bric+ re$ color in$icatin! t%at t%e

microor!anism is a 2-butane$iol pro$ucer. 5-nap%t%ol (G ,) is use$ to intensi"y t%e re$ color by cataly8in! t%e oxi$ation o" */ by

HO: (G,,).

o Enterobacter is G positive.

C. #atalase

o Tests "or t%e presence o" catalase w%ic% converts %y$ro!en peroxi$e to water an$ oxy!en.

2 :2O2 2 :2O F O2

o :y$ro!en peroxi$e is pro$uce$ $urin! oxy!en utili8ation an$ must t%ere"ore be eliminate$ since

%y$ro!en peroxi$e is toxic to t%e cell.

o

3n8yme presence can be teste$ "or by a$$in! I :2O2 to t%e culture an$ loo+in! "or t%e pro$uction o" oxy!en bubbles.

7ubble pro$uction is a positive result.

E. ,?idase

/ytoc%rome c oxi$ase is an en8yme w%ic% can oxi$i8e aromatic amines to "orm a colore$ pro$uct



T%is is also a positive result.

!. ryptone 0+ndole1

o Tests "or t%e en8yme tryptop%anase w%ic% converts trytop%an to in$ole an$ pyruvic aci$.

Tryptop%an pyruvic aci$ F in$ole

o ,n$ole can easily be teste$ "or by a$$in! Hovac@s Dea!ent ( p-$imet%ylaminoben8al$e%y$e (*7*)

an$ :/l $issolve$ in amyl or butyl alco%ol).

o * positive result is w%en t%e Hovac@s rea!ent interacts wit% in$ole.

!!. 7rea

o Tests "or t%e en8yme urease w%ic% converts urea to ammonia an$ carbon $ioxi$e.

Area 2 6: F /O2

o /ontains t%e substrate urea an$ t%e p: in$icator p%enol re$.

o

>%en ammonia is release$ t%e p: o" t%e solution increases an$ once t%e p: is above K.1 t%e p%enolre$ in$icator will appear cerise.

* cerise color in$icates a positive result "or urease.

o Proteus is urease positive.

!2. 3ydrogen Sulfide Production 032S1

o ,t tests "or t%e en8yme cysteine $esul"urase w%ic% removes t%e sul"ur si$e c%ain "rom cysteine to

pro$uce :2& w%ic% w%en in t%e presence o" iron salts (containe$ in Hlin!er@s ,ron *!ar an$ &,

me$ium) "orms a blac+ precipitate.

/ystein :2& F amino acrylic aci$ imino aci$→ pyruvic aci$ F 6:

* blac+ color in$icates a positive result "or cysteine $esul"urase.o Proteus is :2& positive.

!%. S+M

o Tests "or Sul"ur re$uction (:2& pro$uction) presence o" +n$ole an$ Motility.

o :2S positive # blac+ precipitate

7lac+ precipitate is "orme$ w%en t%e pro$uce$ :2& combines wit% iron "rom "errous

ammonium sul"ate "erric sul"i$e (Le&)

o +n$ole positive # Hovac@s rea!ent turns re$ a"ter a$$ition

&ee Tryptone 7rot% above

o Motility positive # !rowt% away "rom inoculation line (appears as clou$iness in tube)!;. Simmons #itrate

o Tests "or t%e ability o" a microor!anism to utili8e citrate as t%e sole carbon source.

oe$ia contains ammonia salts (sole nitro!en source) citrate (sole carbon source) an$ bromot%ymol



b) -cid curd reaction: pin+ soli$ $ue to aci$ pro$uction an$ coa!ulation o" proteins causin! t%e

"ormation o" a soli$.

c) &eduction: litmus is re$uce$ an$ it becomes colorlessM t%e tube appears w%ite since only t%e

mil+ remains. /olor may remain at top o" me$ia w%ere oxy!en can oxi$i8e t%e litmus an$restore its color.

$) -lkaline reaction: appears as a blue liui$ t%at is usually cause$ w%en protein brea+$own

pro$uces amino aci$s t%at are $eaminate$ an$ release ammonia increasin! t%e p: in t%e tube.

e) Peptoniation>Proteolysis: clearin! o" t%e me$ium (may be brown or amber) cause$ byen8yme caseinase w%ic% brea+s $own t%e w%ite protein casein in mil+.

o ore t%an once reaction can be observe$ in a sin!le tube

3xample aci$ cur$ re$uction loo+s li+e aci$ cur$ but t%e tube turns w%ite except "or a small

re!ion at t%e top w%ere oxy!en reoxi$i8es t%e litmus to t%e colore$ (re$) "orm.

!C. 4elatino Tests "or t%e presence o" t%e !elatinase en8yme w%ic% %y$roly8es !elatin to amino aci$s.

o =elatin is a protein t%at soli$i"ies at lower temperatures.

o T%is tube is stab inoculate$ an$ t%en place$ on ice "or several minutes a"ter incubation. *"ter

incubation on ice t%e me$ia may be

iui$ t%is is a !elatinase-positive result (!elatin is %y$roly8e$)

&oli$ t%is is a !elatinase-ne!ative result (!elatin is still present)

*** For the lab midterm you are responsible for knowing everything before this point. PowerPoints, lab lectures,and whiteboards are all fair game for the test. ***

,ther ests>Media:

!E. +MDi#

o&et o" "our tests t%at are use$ to $i""erentiate between Escherichia coli an$ Enterobacter aerogenes

o+n$ol Met%yl De$ Do!es-ros+auer an$ #itrate

+ M Di #

E. coli 8 8 9 9

E. aerogenes 9 9 8 8



2!. HliglerGs +ron -gar 0H+-1

o Tests "or t%e ability to "erment !lucose an$or lactose tests : 2& pro$uction an$ can also be use$ to test

"or !as pro$uction.

/ontains 1I lactose an$ 0.1I !lucose

H,* $i""ers "rom D& $ue to t%e a$$ition o" iron salts to t%e me$ia (see &, above).

o =lucose an$or lactose "ermentation is $etermine$ usin! a p: in$icator (p%enol re$) w%ic% be!ins re$

an$ will turn yellow in t%e butt o" t%e tube i" !lucose is "ermente$. ,t will cause t%e slant to turn yellowi" lactose is able to be "ermente$.

," t%e bacteria contains t%e en8yme cysteine $esul"urase a blac+ precipitant will "orm "rom

t%e reaction o" :2& wit% iron salts.

=as pro$uction can be $etermine$ by crac+s an$or t%e li"tin! o" t%e me$ia o"" t%e bottom o"

t%e tube.

oH,* is i$eally rea$ P1K %ours a"ter inoculation an$ t%e lactose reaction s%oul$ be rea$ "rom t%e bottom

o" t%e slant as t%e tip o" t%e slant may revert bac+ to re$ as t%e inoculation a!es beyon$ 1K-24 %ours in

some species.

22. $ismuth Sulfide -gar 0$S-1

o &tarts o"" as a $ull !reen color.



," plasma becomes clumpy an$ or soli$i"ies t%en bacteria are coa!ulase positive

o Test is only vali$ on =ram-positive stap%ylococcus-li+e bacteria since =ram-ne!ative bacteria are able

to provi$e "alse positive reactions "rom a non-coa!ulase li+e mec%anism.2". Phenol &ed Mannitol Salt -gar 0MS-1

o &elects "or Staphylococcus $ue to %i!% salt concentration ?.5I

o e$ium is re$ but plate an$ colonies will turn yellow i" or!anisms are mannitol-positive.

2C. Staphylococcus !! Medium

o /ontains mannitol an$ ?.5I 6a/l but lac+s p%enol re$ as in &* plate.

o &elects "or Staphylococcus an$ allows "or $evelopment o" natural colony pi!ment "ormation unli+e in

&*.2E. *6ase

o Tests "or t%e exoen8yme 6ase w%ic% is able to %y$roly8e 6* into "ree nucleoti$es.

o et%yl !reen $ye in me$ia complexes wit% intact 6* in t%e me$ia. 7rea+$own o" 6* $isplaces

t%is $ye an$ "orms a clear area.

o Jone o" clearance aroun$ t%e strea+ is a positive result "or t%e presence o" 6ase.

2F. m9Staphylococcus $roth

o 3nric%e$ me$ia containin! 10I 6a/l w%ic% selects "or Staphylococcus since Staphylococcus pre"er

t%e %i!%er salt concentration w%ic% in%ibits most ot%er or!anisms%. (ndo -gar

o &elective "or =ram-ne!ative. /ombination o" so$ium sul"ite an$ t%e basic "uc%sin in%ibits =ram-

positive bacteria.

o i""erential "or lactose "ermentation.

7asic "uc%sin acts as in$icator o" aci$ pro$uction "rom lactose "ermentation.

actose-positive !ives re$ colonies an$ surroun$in! me$ium

o /oli"orms pro$uce a !ol$en metallic !ol$en s%een.



P35S+#-L9#3(M+#-L #,6*++,6S -(#+64 $-#(&+-L 4&,I3

emperature:

*ll species %ave a temperature ran!e "or !rowt% +nown as t%e car$inal temperatures.

• Minimum: lowest temperature permittin! !rowt%

• ,ptimum: best temperature "or !rowt%

• Ma?imum: %i!%est temperature permittin! !rowt%

=roup terms are base$ on t%ese temperature ran!es

• Psychrophiles: reuires low temperatures "or !rowt%.

• Mesophiles: !row in a mo$erate temperature ran!e.

• hermophiles: !row in a %i!% temperature ran!e.

p3:

=roup terms are base$ on p: in !rowt% environment.

• -cidophiles: reuire an aci$ic p: !rowt% ran!e.

• 6eutrophiles: best !rowt% at p: Q.5 to ?.5.

• -lkylophiles>basophiles: reuire al+aline !rowt% con$itions or !rowt% best above p: ?.0.

,?idation9&eduction Potential:

Oxi$ation-re$uction (OD) potential re"ers to t%e presence or absence o" oxy!en. Oxy!en-ric% environments remove

net electrons (aerobic) w%ile oxy!en-$e"icient environment %ave a net increase in electrons (anaerobic). icrobesreuire t%e presence o" oxy!en t%e absence o" oxy!en or tolerate bot% con$itions ("acultative). ,n $eterminin! t%is

trait t%e re$ucin! a!ent t%io!lycollate can remove oxy!en except at t%e topmeniscus. T%us you can $etermine t%e

position o" !rowt% in t%e tube an$ !roup t%e bacteria accor$in!ly. Desa8urin is use$ as a re$ox in$icator colorless inre$ucin! con$itions an$ pin+ w%en oxi$i8e$.

=roup terms are base$ on OD reuirements or tolerance.

• Strict aerobes: reuires oxy!en "or metabolism an$ maintenance o" cell structure. 7acteria !row only at

t%e top o" t%e tube.

• acultative species: can !row wit% or wit%out oxy!en. 7acteria !row t%rou!%out t%e tube.

• Strict anaerobes: inactivate$ by oxy!en exposure (metabolism is "ermentative) cell structure may bealtere$ by oxy!en. 7acteria !row only at t%e bottom o" t%e tube.

,smotic Pressure 0aw1:

Min ,pt Ma?

Psychrophile 0R/ 5R/ 15-20R/

Mesophile 20R/ 0-?R/ 45R/

hermophile 45R/ 55-Q5R/ ?0-S0R/



4&-M P,S++D( P5,4(6+# #,##+

e$ical icrobiolo!y is primarily concerne$ wit% t%e isolation an$ i$enti"ication o" pat%o!enic or!anisms. One

o" t%e most "reuently encountere$ types o" pat%o!enic bacteria are t%e =ram-positive cocci. O" t%ese bacteria t%etwo !enera we will "ocus on are Staphylococcus an$ Streptococcus .

taphylococcus

Loun$ in nasal membranes t%e %air "ollicles t%e s+in an$ perineum.

ost strains are penicillin resistant w%ic% can cause epi$emiolo!y problems since S0I o"

%ealt%care wor+ers carry Staphylococcus.

ivi$e in multiple planes an$ t%ere appears as irre!ular clusters microscopically.

T%ree important Staphylococcus species are S. aureus, S. epidermidis, and S. saprophyticus.

o O" t%ese only S. aureus %as t%e ability to coa!ulate plasma.

treptococcus Loun$ in p%arynx on sur"aces o" t%e teet% saliva s%in colon rectum an$ va!ina.

ivi$e in only one plain an$ t%ere"ore appear as c%ains o" cocci.

Streptococci o" !reatest me$ical si!ni"icance are S. pyogenes, S.pneumoniae, and S. agalactiae.

+solation and +dentification of Staphylococcus and Streptococcus:

*ay !:

&wab nose (6) t%roat (T) an$ a "omite (L).

&trea+ swab onto 7loo$ *!ar plate as s%own.

lace swabs into m-&tap%ylococcus 7rot%.

*ay 2:

taphylococcus!

o Ase t%e t%ree m-&tap%ylococcus 7rot%s to inoculate two &tap%ylococcus e$ium 110

(&110) an$ two annitol &alt *!ar (&*) plates.o &elect a beta-%emolytic Staphylococcus "rom t%e 7loo$ *!ar (7*) plate an$ inoculate a

t%ir$ & 110 an$ &* plate.

treptococcus!



T%is will be your positive control.

*ay ;:

taphylococcus!

o 3xamine t%e results o" t%e 6itrate 7rot% 6ase an$ /oa!ulase inoculations ma$e last

perio$ an$ i$enti"y t%e species o" Staphylococcus. treptococcus!s

o 3xamine t%e results o" t%e 6itrate 7rot% inoculations ma$e last lab an$ i$enti"y t%e

species o" Streptococcus .



4&-M 6(4-+D( +6(S+6-L P-3,4(6S

=ram-ne!ative intestinal pat%o!ens are a major concern "or public %ealt% since t%e two main

pat%o!ens Salmonella an$ Shigella %ave t%e ability to cause enteric "evers "oo$ poisonin! $ysentery an$even typ%oi$ "ever. Salmonella %as over 2200 serotypes inclu$in! Salmonella typhi t%e cause o" typ%oi$

"ever an$ Shigella contains many serotypes some o" w%ic% are t%e main cause o" %uman $ysentery.

ublic :ealt% aboratories routinely test "or t%e presence o" t%e =ram-ne!ative pat%o!ens by t%e

isolation an$ i$enti"ication o" Salmonella an$ Shigella "rom "eces. T%is ma+es isolation an$ i$enti"ication$i""icult $ue to t%e presence o" Escherichia Proteus Enterobactor Pseudomonas an$ Clostridium in "eces

(t%ere are actually between 00 to 1000 $i""erent species o" bacteria in t%e !ut %owever only 0 to 40 o"

t%ose species ma+e up t%e majority o" t%e bacteria "oun$ in t%e !ut). ,n t%is lab we will isolate an$ i$enti"y

Salmonella "rom a "eces-li+e mixture o" bacteria.

&ome i$enti"yin! traits o" Salmonella are =ram-ne!ative ro$ lactose-ne!ative motility-positive :2&-

positive urease-ne!ative an$ t%e "ormation o" small colonies on a!ar.

*ay !:

er"orm isolation strea+s o" t%e Salmonella containin! mixture onto eac% o" t%e

selective$i""erential me$ia provi$e$.

*ay 2:

Lrom t%e "ive selective$i""erential plates select seven isolate$ colonies t%at are presumptive"or bein! Salmonella base$ on t%eir appearance an$ inoculate eac% into a &, Area an$ H,*

me$ia.

o /olonies to be selecte$

(M$: lactose-ne!ative bacteria (colonies $o not c%an!e color)

*(S #itrate: lactose-ne!ative bacteria (colonies $o not c%an!e color)

$4-: lactose-ne!ative (colonies appear pin+w%ite surroun$e$ by re$ me$ia)

SS -gar: blac+ colonies

$S-: blac+ colonies

*ay %:

etermine t%e i$entity o" eac% o" t%e selecte$ colonies.



S-6*-&* PL-( #,76

To $etermine a bacterial population count (t%e number o" or!anisms t%at are present in a !iven unit

o" volume) several met%o$s are available t%e one o" t%e simplest met%o$ bein! t%e stan$ar$ plate count.T%e stan$ar$ plate count is accomplis%e$ by $ilutin! t%e culture sample an$ !rowin! t%e bacteria up on

6utrient *!ar. /ountin! t%e number o" colonies t%at !row on 6utrient *!ar $etermines t%e number o"

bacteria in t%e $ilute$ sample. To $etermine t%e bacterial population count o" t%e ori!inal culture one ta+es

t%e bacterial count in t%e $ilute$ sample an$ multiples by t%e $ilution "actor.*$vanta!es o" t%e &tan$ar$ late /ount over ot%er 7acterial late /ount et%o$s is t%e "act t%at it

%as a very basic principle an$ tec%niue t%at reuires very minimal amount o" euipment but still provi$es

excellent results. isa$vanta!es o" t%e &tan$ar$ late /ount are t%at t%is met%o$ can only count bacteria

w%ic% are able to $evelop un$er t%e !rowin! con$itions provi$e$. T%ermop%iles or $ea$ colonies cannot be

counte$ usin! t%is proce$ure.

&ules for Standard Plate #ount:

1. ic+ t%e plate t%at contains between 0 ' 00 bacterial colonies to count.2. /alculate all t%e $ilution "actors (L) between t%e counte$ plate an$ t%e ori!inal culture.

a. L # amount a$$e$ (amount a$$e$ F amount alrea$y t%ere)

• * ->0-8$1

b. L "or platin! # amounte$ a$$e$ 1ml stan$ar$

• Plated * ->!ml

. >%en !oin! "rom t%e counte$ plate bac+ towar$s t%e ori!inal culture multiple t%e plate count by t%einverse o" all t%e $ilution "actors.

(?ample:

1. &elect plate wit% 2 colonies

2. late$ $ilution # 1ml1ml # 1

*ll ot%er $ilutions # 1(1FSS) # 1100

. 7acterial plate count # 2 x 1 x 100 x100 x 100 # .2 x 10? bacteriaml



M5#,L,45

erminology:

• ycolo!y is t%e stu$y o" "un!i ( p) ("un!us s).

• N Myco- or N-mycete in$icates "un!i or similarity to "un!i.

#ellular raits of ungi:

• T%ey are nonmotile eu+aryotes t%at possess nuclei an$ or!anelles.

• Lun!i seem plant-li+e but are not plantsM t%ey $o not possess c%loroplasts an$ are not

p%otosynt%etic.

•

ar!e cells ( 10Um) ma+in! t%em visible usin! low ma!ni"ication (10V objective).• /ontain a simple cell wall o" chitin (polysacc%ari$e) not similar to t%e complex pepti$o!lycan o"

bacterial cells or cellulose cell wall o" plants.

• Lun!i are absorptive heterotrophs t%ey release exoen8ymes into t%e environment an$ t%en

absorb an$ $i!est t%e nutrients.

• ost are saprophytes t%at $ecompose $ea$ an$ $ecayin! or!anic matterM "ew are parasites o"

plants animals an$ %umans.

• ost "un!i are aerobicM "ew are "acultative or aerotolerant anaerobes.

• ,n"ormally $ivi$e$ into

a. Lilamentous mol$s t%at create multicellular %yp%ae. ,t also inclu$es macro"un!i w%ic% pro$uce "les%y repro$uctive structures (pu""balls mus%rooms an$ s%el" "un!i).

b. Anicellular yeast t%at repro$uce by bu$$in!.

c. imorp%ic "un!i w%ic% %ave bot% mol$ (25R/) an$ yeast (?R/) li"e-cycle sta!es.

#ultural raits of ungi:

• Lun!i tolerate a broa$ ran!e o" p: an$ osmotic pressures.

• :ave a narrower tolerance o" temperature an$ re$ox con$itions.

• e$ia selective "or "un!i will %ave an aci$ic p: between 4.K to 5.0.• *ble to metaboli8e complex substrates inclu$in! woo$y wastes suc% as cellulose li!nin tannins

an$ %emicelluloses.

o Lun!i are critical "or $ecomposition o" "orest litter an$ $ea$ trees (saprop%ytes).



• &ome yeast can pro$uce pseudohyphae.

Properties of ilamentous ungi 0molds1:

• /%aracteri8e$ by t%e $evelopment o" hyphae ( p) are in$ivi$ual "un!al "ilaments (hypha s).

o &eptate$ %yp%ae contain cross-walls "ormin! multiple compartments in t%e %yp%a.

o 6onseptate %yp%ae eac% in$ivi$ual %yp%a is a sin!le uninterrupte$ compartment.

o ycelium colonial mass o" all t%e %yp%ae.

o &ome mol$s also pro$uce a sac-li+e cell +nown as sporangia.

o any "un!i (suc% as Mucor spp.) "orm complementary (F) an$ (-) matin! types bot% o"

w%ic% are reuire$ "or sexual repro$uction.

o T%ose t%at "orm "les%y repro$uctive structures (mus%room pu""balls an$ s%el" "un!i) are

+nown as macrofungi.o * portion o" t%e mycelium !rows into t%e nutrient supply an$ is re"erre$ to as t%e

substrate mycelium.

o art o" t%e mycelium projects upwar$ to pro$uce an$ sprea$ sexual an$ asexual spores.

T%ese are +nown as aerial mycelium.o $lastospores are asexual spores pro$uce$ by bu$$in!.

4roup erms for ungi:

T%eir !roupin! is base$ on sexual repro$uction an$ sexual spores as well as in t%e presence o" cross walls

in t%e %yp%ae. Se?ual spores occur a"ter meiosis

Jy!ospores spores locate$ in a 8y!osporan!ia unenclose$ spores.

*scospores spores locate$ in an ascus (sac-li+e) cell.

7asi$iospore spores locate$ in a basi$ia (a microscopic spore "ormin! structure). -se?ual spores are "orme$ in t%e sporan!ium a"ter mitosis an$ cytoplasmic cleava!e

&poran!iospores enclose$ in sporan!ium sac.

/oni$iospores unenclose$ expose$ spores "orms conidia at t%e en$ o" %yp%ae.

+. Kygomycetes• Ge!etative %yp%ae "ilaments are branc%e$ an$ nonseptate.

• Jy!ote un$er!oes meiosis an$ pro$uce a sexual ygospores.

• /an also pro$uce sporangiospores (asexual spores) in a sporan!ium locate$ on t%e apical en$ o"



+++. $asidiomycetes

• &eptate %yp%ae t%at are compacte$ into lar!e "ruitin! bo$ies (mus%rooms s%el" "un!i pu""balls

etc).

+D. *euteromycetes

• *lso +nown as N"un!i imper"ecti.

• 6early in$istin!uis%able "rom septate coni$iospore-"ormin! ascomycetes.

• T%is !roup is %as never been scienti"ically proven to %ave a sexual repro$uctive sta!e.

• &exual repro$uction in t%is p%yla is still consi$ere$ possible just unproven.



$-#(&+-L (-M+6-+,6 , I-(&

>ater is routinely examine$ "or "ecal contamination to ensure sanitary water supplies. Lecal

contamination is i$enti"ie$ by "in$in! coli"orms in t%e water. * coli"orm is $e"ine$ as a "acultative anaerobet%at "erments lactose to pro$uce !as an$ is a =ram-ne!ative non-spore-"ormin! ro$. Two bacteria t%at "it

t%is $escription are Escherichia coli an$ Enterobacter aerogenes. &ince E. coli an$ E. aerogenes bot% "it

t%e $escription o" coli"orms an$ E. coli is a better in$icator o" sewa!e contamination t%an E. aerogenes t%e

,Gi/ tests must be per"orme$.

ualitative ests:

!. Presumptive est 0*ay !1:

o * series o" S-12 tubes o" lactose brot% use$ to i$enti"y i" t%ere are any bacteria in t%e water t%at

are lactose "ermentin! !as pro$ucin!. T%ese tests are also use$ to $etermine t%e most probablenumber (6) o" coli"orms present per 100 ml o" water.

T%e 6 is $etermine$ by observin! t%e number o" lactose brot%s tubes t%at are

positive "or !as (positive result # 10I or more o" ur%am tube "ille$ wit% !as) an$

comparin! t%ose number to t%e 6 etermination Table.2. #onfirmed est 0*ay 21:

o 37 or 3n$o *!ar plates are inoculate$ "rom !as positive lactose brot%s. On 37 a!ar

coli"orms pro$uce small colonies wit% $ar+ centers. On 3n$o a!ar coli"orms pro$uce re$$is%

colonies. T%ese results in$icate t%at t%e i$enti"ie$ colonies are =ram-ne!ative lactose-positive

bacteria. On bot% 37 an$ 3n$o a!ars metallic s%een is also a result o" =ram-ne!ativelactose-positive bacteria. T%is step is necessary because in real-worl$ samples you will %ave a

mix o" bacteria an$ you will nee$ to select "or a lactose-positive colony to "urt%er tests to

$etermine i" it is E.coli t%at cause$ t%e positive !as reaction or somet%in! else.%. #ompleted est 0*ay %1:

o actose-positive =ram-ne!ative colonies are selecte$ an$ inoculate$ into lactose brot% an$

onto 6utrient *!ar slants. ," !as is pro$uce$ in t%e lactose brot% an$ stainin! reveals t%at t%e

bacteria are =ram-ne!ative ro$s an$ non-spore-"ormin! t%en t%e water is $etermine$ to be positive "or t%e presence o" coli"orms.

;. +MDi#:o To con"irm t%at t%e positive coli"orm test is $ue to E. coli an$ not E. aerogenes t%e ,Gi/ set

o" tests must be con$ucte$.

M b ilt M th d



-6+S(P+# (D-L7-+,6

!. ilter Paper *isk Method

o Ase$ to compare antiseptics base$ on t%eir bacteriostatic properties.

o Delative e""ectiveness is measure$ by t%e si8e o" t%e 8one o" in%ibition (measure$ "rom e$!e o"

plate to e$!e o" in%ibition 8one) an$ can be compare$ uantitatively a!ainst ot%er substances.

o easures t%e relative e""ectiveness o" t%ree a!ents (p%enol "ormal$e%y$e an$ io$ine) a!ainst

two or!anisms (Staphylococcus an$ Pseudomomas).2. Hirby9$auer Method

o Ase$ to compare e""ectiveness o" antiseptics bot% antibiotics (ma$e naturally) an$ $ru!s (man

ma$e) w%ic% is $one un$er a stan$ar$i8e$ system.

o et%o$olo!y stan$ar$i8es $i""usibility o" t%e a!ent si8e o" t%e inoculum type o" me$ium an$

many ot%er "actors.

o Delative e""ectiveness is measure$ by measurin! t%e $iameter o" t%e 8ones o" in%ibition.

%. Minimum +nhibitory #oncentration 0M+#1

o 3stablis% t%e minimum concentration o" an antiseptic nee$e$ to in%ibit t%e !rowt% o" a test

microor!anism.o ,/ proce$ures

a+e $ilutions o" t%e antiseptic nee$e$ "or testin! as s%own



76H6,I6S

Bour un+nown will contain 2 $i""erent or!anisms "rom t%e "ollowin! list

cinetobacter

lcaligenes

Bacillus

Corynebacterium

Enterobacter

Eschericia

$lebsiella

%actobacillus

Micrococcus

Mora&ella

Mycobacterium

'eisseria

Proteus

Pseudomonas

Salmonella

Shigella

Staphylococcus

Streptococcus

Procedure:

1. Decor$ t%e number o" your un+nown2. =ram-stain your un+nown.

• Decor$ =ram@s result s%ape an$ arran!ement o" t%e or!anisms in your un+nown.

. &trea+ your un+nown mixture onto 2 T&* plates an$ 2 7:, plates. lace 1 T&* an$ 7:, plate int%e incubator an$ t%e ot%er set o" plates at room temperature.

4. ,$enti"y isolate$ colonies t%at appear $i""erent on t%e isolation strea+s.

•

=ram-stain to ensure isolation an$ i$enti"ication.5. Trans"er eac% $i""erent isolate$ colony onto 2 T&* an$ 2 7:, slants.

• 1 T&* an$ 1 7:, slant will be your wor+in! stoc+ w%ile 1 T&* an$ 1 7:, will be your

reserve stoc+.

• >%en you use a slant "or inoculation $iscar$ t%at slant an$ reinoculate a "res% slant "rom

t%e reserve stoc+. T%e ol$ reserve stoc+ will be come your wor+in! stoc+ "or t%e next lab

an$ t%e newly inoculate$ slant will become t%e reserve stoc+.

Q. Asin! t%e "ollowin! "lowc%arts all ot%er materials provi$e$ t%rou!%out t%is class an$ 7er!ey@s

anual (De"erence &ection o" t%e ibrary) $etermine t%e i$entity o" your un+nowns.

?. &ubmit your results usin! t%e $ic%otomous +ey on /anvas. ,nclu$e your un+nown number. /irclet%e or!anisms you "in$ an$ t%en (usin! a %i!%li!%ter) %i!%li!%t t%e pat% you too+ to "i!ure out

your or!anisms.

#ommon (rrors Ihen Iorking with 7nknowns:



76*(&S-6*+64 -6* +6(&P&(+64 &(S7LS +6 M(*+- , *+(&(6+-( $-#(&+-L SP(#+(S

MEDIUMTYP

EOBJECTIVE

SIGNIFICANTCOMPONENT

SINDICATOR RESULTS

PhenylethanolAgar PEA!

Selective

Selects for Gram-positive

Phenylethanol(PEA) inhibitsGram-negativegrowth

Ability to grow with PEAGro"th = Gram-positiveNo gro"th = Gram-negative

Eo#$n MethyleneBl%e Agar EMB!

Selective

&Differenti

al

Selective for Gram-negativeDifferential for actosefermenters

actose! eosin!an" methylenebl#e "ye

$olonies absorb "yes "#e tothe low p% ca#se" fromlactose fermentation

Dar& 'olony = lactose fermenter ()!absorbs ' "#e to high aci"pro"#ctionP$n& 'olony = lactose fermenter ()!absorbs eosin "#e to low aci"pro"#ction

No (ye a)#or)e( = lactose fermenter(-)

De#o*y'holateAgar DES!

Selective

&Differenti

al

Selective for Gram-negativeDifferential for actosefermenters

actose! an""esoycholate("etergent)

*e" "ye is absorbe" intoaci" pro"#cing colony "#ringlactose fermentation

Re( 'olony = lactose fermenter ()Non+re( 'olony = lactose fermenter (-)

Bloo( Agar Differenti

al

Detects hemolysins byobserving hemolysis

+, sheep re"bloo" cells(*$)

Action on *$

Al,ha he-oly#$# = partial lysis of *$Beta he-oly#$# = complete lysis of*$ Ga--a he-oly#$# = no lysisof *$

Star'h Agar Differenti

al

Detects amylase whichhy"rolyes starch tosimple s#gar

Sol#ble starch.o"ine is a""e" after growth.o"ine starch = p#rple

Colorle## aro%n( 'olony = amylase()P%r,le all aro%n( 'olony = amylase(-)



L$,a#e Agar Differenti

al

Detects lipase whichhy"rolyes lipi"s to fattyaci"s an" glycerol

$orn oil lipi" /e#tral bl#e "ye

Bl%e (ye $nten#$.$e# = hy"rolase ()!color "#e to fatty aci" release loweringthe p% No)l%e $nten#$.$'at$on = hy"rolase (-)

M$l& Agar Differenti

al

Detects caseinase whichhy"rolyes casein! a mil0protein

'il0 (containing

opa1#e casein)oss or retention of opacity

Clear$ng o. o,a/%e -$l& = caseinase() No 'lear$ng o. -$l& = caseinase (-)

Gelat$nDifferenti

al

Detects gelatinase whichhy"rolyes gelatin proteininto AA

Gelatin*esoli"ification of gelatin atlower temperat#re

Re#ol$($.$'at$on = gelatinase (-)No re#ol$($.$'at$on = gelatinase ()

Try,tone BrothDifferenti

al

Detects tryptophanasewhich hy"rolyestryptone to in"ole 2ests

for presence of in"ole

2ryptonepolypepti"econtaining

tryptophan

3ovac4s reagent is a""e"after growth (5-+ "rops)

Re( to, = tryptophanase () Yello" to, = tryptophanase (-)

N$trate BrothDifferenti

al

Detects nitrate re"#ctase(nitrase) which convertsnitrate to nitrite

So"i#m nitrate

After growth a"" /itrate . &.. (67 "rops each) an" inc"#stoo0 for a re" comple toform

Re( "$tho%t 0n = nitrase ()Re( "$th 0n = nitrase (-)Ne1er t%rn# re( = nitrase ()

Mot$l$ty Me($aDifferenti

al

Determines if the

bacteria is motile

2etraoli#mchlori"e

(growthin"icator)

2etraoli#m chlori"e8

re"=bacteria growth

Re( thro%gho%t t%)e = motility ()

Re( only near #ta) = motility (-)

L$t-%# M$l& BrothDifferenti

al

Detects mo"erate9heavyaci" from lactosefermentation! presence of caseinase an" re"#ctase

actose!itm#s! caseinfrom mil0

Aolitmin "ye (itm#s-oi"ie" form)8laven"er=ne#tral! pin0=aci"!bl#e=al0aline! nocolor=re"#ce" form

+ to : reactions can be rea" in itm#s'il0 (see below);;



;;(6) mo"erate aci" pro"#ction from lactose = pin0! li1#i" in t#be< () heavy aci" pro"#ction from lactose = pin0! har" or soft c#r"s (coag#lation of casein by aci")<(5) al0aline pro"#cts from metabolism = bl#e! li1#i"< (>) re"#ction of litm#s "ye to colorless in lower half of broth = viewe" as whitish from nat#ral mil0 color when the litm#s"ye is no longer in the oi"ie" colore" state? D#e to bacterial re"#ctase in mil0< (+) loss of opa1#e casein protein by caseinase = proteolysis (peptoniation)!#s#ally seen starting at the top an" increases "ownwar"< (:) slimy stran"s s#spen"e" in li1#i" state of me"i#m = ropiness trait pro"#ce" by caps#le-pro"#cing bacteria



MEDIUM TYPE OBJECTIVE SIGNIFICANT COMPONENTS INDICATOR RESULTS

Urea Broth DifferentialDetects #rease ("egra"es #rea toammonia an" carbon "ioi"e)

@reaPhenol re"8 yellow=aci"!re"=ne#tral! cerise=al0aline

Re( to 'er$#e = #rease () Yello" to l$ght orange = #rease (-)

Methyl Re(MR!

DifferentialDetect mie" aci" fermenters(heavy aci" pro"#ction)

Gl#cose'ethyl re" is a""e" after growth(5-+ "rops)! a p% below +?6=re"

Dye #tay# re( = mie" aci" fermenter ()T%rn# yello" = mie" aci" fermenter (-)

Voge#+Pro#&a%er VP!

Differential Detect !5-b#tane"iol fermenters Gl#coseP . & .. are a""e" after growth(67 "rops each)

Re( on to, = !5-b#tane"iol fermenter ()No re( 'olor = !5-b#tane"iol fermenter (-)

S$--on#

C$trate Slant Differential

Detect #se of citrate as sole

carbon so#rce $itrate

romothymol bl#e8 yellow=aci"!

green=ne#tral! bl#e=al0aline

Gro"th on #lant an( )l%e 'olor = ()

No gro"th = (-)Phenol Re(PR! S%garFer-entat$on

DifferentialDetects fermentation of selecte"s#gar Detects pro"#ction of gas

S#gar (variable) an" D#rhamt#be

Phenol re"8 yellow=aci"!re"=ne#tral! cerise=al0aline

Yello" = fermenter ()L$ght orange to re( = fermenter (-)Ga# $n t%)e = gas pro"#ction ()

2l$gler3# Iron2IA! T%)e

DifferentialDetects fermentation of gl#cose!lactose! an" %S pro"#ction

Gl#cose (7?6,)! lactose (6?7,)an" iron salts

.ron ions precipitate %SPhenol re"8 yellow=aci"!re"=ne#tral! cerise=al0aline

Re( #lant4yello" )%tt = gl#cose fermenterAll yello" = gl#cose & lactose fermenterBla'& = %S pro"#ction

Phenylalan$ne Differential

Detects phenylalanase(phenylalanine "eaminase) whichconverts phenylananine tophenylpyr#vic aci" (PPA)

Phenylalanine (amino aci")Berric chlori"e is a""e" aftergrowth (+-67 "rops)

Green = phenylalanase ()No 'olor 'hange o. .err$' 'hlor$(e =phenylalanase (-)

O*$(a#e Differential

Detects oi"ase which removes

hy"rogen from "ifferents#bstrates

Dimethyl- p-phenylene"iamine

hy"rochlori"e (oi"ase reagent)

Dimethyl- p-phenylene"iaminehy"rochlori"e is a""e" after

growth! "ar0 re"9p#rple to blac0 =oi"ie"

Dar& re(4,%r,le to )la'& = oi"ase ()

Re-a$n 'olorle## = oi"ase (-)

Catala#e DifferentialDetects catalase which convertshy"rogen peroi"e to water an"oygen

5, hy"rogen peroi"e is a""e"after growth

B%))le# = catalase ()No )%))le# = catalase (-)

#(LL7L-& S-+6+64

STAINING TYPE REASON FOR STAININGPRIMARY

STAINMORDANT

DECOLORI0ER

SECONDARYSTAIN

RESULTS

Negat$1e Simple.nsol#ble negative "ye stainsthe bac0gro#n"

.n"ian in0 or/igrosin

ac0gro#n" stains blac0! bacteria aretransparent

S$-,le SimplePositively charge" stain!negatively charge" bacteria

'ethylenebl#e

All bacteria stain bl#e

Gra- Differential Differences in cell wall layers $rystal violet .o"ine Ethyl-alcohol SafraninP%r,le = Gram ()Re( = Gram (-)

A'$( Fa#t DifferentialDifferences in way content ofcell wall

$arbolf#chsin

(%eat) Aci"-alcohol 'ethylene bl#eRe( = Aci"-Bast ()Bl%e = Aci"-Bast (-)

S,ore Str#ct#ral%eat "riven "ye is retaine" byspores

'alachitegreen

(%eat) Cater SafraninGreen = spores9en"osporesRe( = all other cell#lar material

micro lab packet updated january 2016

Documents