Chapter 10

Methodology Materials: a. Media Eosin-Methylene Blue Agar (EMB) Mannitol Salt Agar (MSA) Nutrient Ag Agar (N (NA) l lates an and sl slants !lucose "er#entation Broth (!"B) Si##ons Citrate Agar (SCA) NB $ !elatin *ee Lit#us Milk Motility Indole ,rnithine Agar (MI,) Nutrient Broth

Lysine Iron Agar (LIA) MacConkey Agar (Mac) Starch Ag Agar (SA (SA)) l late NB $ %.&' glucose rytone Broth rile Sugar Iron Ag Agar(SIA) Butt+Slant Nitrate Broth rea Agar late SA slant

/. Culture

Acronym S.A. S.C. E.C. 0.,. .. S.M. E.A. B.S.

Scientifc Name Staphylococcus Staphylococcus aureus Staphylococcus cohnii Escherichia coli Klebsciella oxytoca Pseudomonas putida Seratia marcescens Enterobacter aerogenes Bacillus subtilis

c. 1eagents 2' 3ydrogen ero4ide Methyl 1ed Indicator 0o5acs reagent Nitrate reagents A7B Mineral oil

!ra# Iodine Solution &' alha-nahtol 6%' 0,3 Saturated Coer (II) Sul8ate (CuSo 6) solution

Main rocedure: 9 di;erent #edia+/roth+slants
In #aking= co#onents o8 e5ery #edia are added one /y one as stated in the aendi4 8ro# the Micro/iology la/oratory #anual. reared #edia ected to autocla5ing /e8ore inoculation. Autocla5ed #edia ected to di;erent /ioche#ical tests a8ter seci?c cultures
3al8 o8 the reared slants= dees= /utt-slants and lates
1esults
Bacteria A. !ro
,ne MSA late
 )hese
Colonies
. Eosin-Methylene Blue Agar

,ne MSA lates
 )hese
2. MacConkey Agar

Colonies
,ne MSA lates
 )hese
Colonies
B. est 8or Catalase 9. Catalase est NA slants and lates
9- dros o8 2' 3ydrogen ero4ide
 )h e e5olution o8 /u//les o8 o4ygen gas
C. Meta/olis# o8 Car/ohydrates and 1elated Co#ounds 9. Starch 3ydrolysis est

A streak o8 Bacillus It
"looded
1esults
. !lucose "er#entation /y In5erted u/e ( *urha# u/e)

 )h e set-u used
,ne tu/e
 )h ese
Acid and gas roduction
2. Methyl 1ed (M1) and oges-roskauer () ests 8or the roduction o8 acids and Acetyl#ethylcar/inol (AMC) or Acetoin

,ne M1 tu/e and one  tu/e
A8ter incu/atrion= hal8 o8 the culture
AMC test
Acid roduction
4. Citrate Utilization est  , n e S C A s la n t < a s inoculated
 ) h e  re s e n c e o8 gro
 ) h e s e < e re incu/ated at 2@ * eg. Celsius 8or 6: hours.

&. Sugar tiliation and 3 S roduction sing rile Sugar Iron Agar

,ne )SIA tu/e
 )hese
1esults
!as roduction = trace o8 /lackening= and /lack reciitate
*. Meta/olis# o8 Nitrogen-Containing Co#ounds 9. !elatin 3ydrolysis est ,ne tu/e
 )hese
. est 8or Indole roduction

 )he hydrolyse d gelatin
,ne tu/e
& dros 0o5ac(s reagent
No #i4ing
1esults
2. 1eaction 8or Lit#us Milk ,ne tu/e
 )hese
1eactions
6. Nitrate 1eduction est

,ne tu/e
9#l o8 nitrate reagent A
A inch o8 inc o
A distinct red color
&. Lysine *ecar/o4ylation and *ea#ination ests LIA tu/es
 )hese
ossi/le results
G. rease est (Stuarts rea Broth Method)

rease test
A hea5y inoculu#
 )u/es
Color change
@. Motility Indole ,rnithin Agar *ee MI, dees
 )hese
ossi/le results
1e8erences Bahra#i= M.= et al. (%96). hysiological Characteriation o8 Microorganis#s. !aboratory "anual in #eneral "icrobiology.   -H.

Microbial reactions in culture media (summarize page 138- 141 (san to p!ede ilagay" #iscussion"