MICNOBIOLOGT - PLATE NEADING

ROUTT NE CU LTURE MEDIA/MICROSCOPIC AN D

MACROSCOPIC MORPHOLOGY depressed center/gray mucoid

media
.{- GRAM POSITIVE ORGANISMS:
,/ staphylococcus species
. Gram stain: Gram (+)cocci in clusters
. White colonies - CONS
r B-whitelB-yellow - S. aureus
2. Viridans group

ir,i.,,*Offi
j'Jtf i
' ..& r.l'']'
. Biochemicaltest:
)Coagulase (+)* S. aureus
)Coagulase (-)- CONS
FCatalase (+)- CONS & S. aureus
,/ Micrococcus species
. Gram stain: Gram (+) cocci in tetrads
. White colonies I Gamma hemolytic
, Biochemical test:
r' Streptacoccusspecies
, Gram stain: Gram (+) cocci in chains;
catalase (-)
, 6-hernolyticStreptococcus
1. Streptococcus Pyogenes (GrP A)

'.'!
+ GRAM NEGATTVE oRGAN|SM {BAP}
r' Gram stain: Gram (-) bacilli
2. Streptococcus aga ,/ Morphology:
' Gray mucoid - Klebsiella
' GrayirregularfeatherY
edge/serrated - Pseudomonas
r GraY swarming - Proteus
' Gray flat - Escherichia coli
' GraY small-
I a- h e m o lyt ic Stre pto coccu s
1. Streptococcus pneumoniae
r Taxo P (Optochin - S)
 MICBOBIOLOGY - PLATE BEADING

+ YEAST (staphylococcus species) and differentiol {5.

./ Gram stain: yeast aureus & CONS)
,/ Morphology: ,/ CHO: Mannitol
. Cottony white ,/ lnhibitor: 7.5-10% NaCi
. White spike ,/ pH indicator: Phenol Red

' Gray dull ,/ mannitol fermenter - yellow (S.

. White with feet aureus)
,/ non-mannitol fermenter - pink (CONS-
S. epidermidis and S. saprophyticus)
o Biochemical test: Novobiocin
Resistant Test:
r R-S.saprophyticus
+ Corynebacterium diphtheriae r $*S.epidermidis
,/ Gram stain: Gram (+) bacilli (Chinese letter)
./ Morphology:dullgray ,/ Almost all organism + fastidious
t' organism ( Haemophilus and Neisseria)
'/ Cell lysis provides for the release of
,t intracellular nutrients such as hemin (X
factor) and coenzyme nicotinamide
adenine dinucieotide (NAD or V factor)
into the agar for the utilization of
negative bacilli ond differential$actose and fastidious organism
nan -la ctose fe rme nter) '/ Haemophilus and Neisseria -
/ CHO: Lactose translucent gray
,/ lnhibitor: Crystal Violet and Biles Salts
For H. influenzae
(inhibit gram (+) and yeast)
/ pH lndicator: Neutralred BAP (X Factor) + S. aureus (V factor) = trans
{ Lactose fermenter - pink gray (H. influenzae) "satellitism"
r' Non-lactose fermenter - colorless

{ E. coli- LF with bile precipitate

'/ ""f "fi::;::'l'J*:'$""'

' brown
PYomelanin -
/ "I'"fi#il#,*,:,,:ffi:
r' Acinetobacter - purple hue
,/ Serratia marcescens - red pigment
(prodigiosin)
,/ Chromobacter violaceum - purple
swarming of Proteus
pigment
r' For isolation of Neisseria species (trans
gray)
 MICROBIOLOGY - PLATE READING

'/ naturally rich medium used for the

'/ Bacitracin -
selective agent which is cultivation of fastidious and
inhibitory to most strains of nonfastidious microorga n isms,
streptococci, staphylococci, Neisseria including aerobic and anaerobic
spp and Micrococcus spp. bacteria
'/ For selective isolation of Haemphilus '/ for sterile body fluids (csf, peritoneal,
influenzoe f rorn respiratory specimen pericardial, synovial, pleural, ascitic
fluid)
,/ Gentamicin: selective agent inhibitory
to most Enterobacterales, Neisseria, '/ supports the growth of aerobes,
and Staphylococcus species. microaerophiles, anaerobes and
'/ selective isolation of Streptococcus fastidious organism
pneumoniae r' resazurin = oxidation-reduction
', THIOSULFATE CITRATE BILE SALTS SUCROSE indicator
AGAR (rCBS) / thioglycolic acid = acts as a reducing
'/ H25 indicator: Ferric citrate and agent to create an anaerobic
sodium thiosulfate environment deeper in the tube, allow
'/ Gram (+) and Gram (-) rods inhibitor: anaerobic bacteria to grow

./
Na citrate, Bile salts and Na thiosulfate '/ *
",Y*:;i,'ff ::,1 3f,l::?,,as,
i ra tes

pH indicator: Bromthymol Blue

(alkaline -green; yellow)
361fl =
IIt
./ Sucrose fermenter = yellow colonies
' e.g: Vibrio cholera (large flat il
yellow) and Vibrio alginolyticus LI
atlLg tto Obi,!,!o i!rcro fi.,rll.rlrr'.,
,/ NOn- Sucrose fermenter = gr€€11
A.r..L! .'n raroblr noroDhtl+ r.iJ.robt

colonies + SELENITE F
. e.g: Vibrio parahaemolyticus r' enhance the growth of Salmonella
(large flat green) and Shigella
r' contains sodium hydrogen selenite
,/ Selective - differential for Salmonella which is toxic for most
and Shigella Enterobacterales
,/ cHo: Lactose + ATKALINE PEPTONE WATER
./ lnhibitor: brilliant green and bile salts ./ Used to enhance the growth of
./ pH indicator ; Neutral Red Vibrio
./ H25 indicator: Ferric Citrate
. Saimonella - NLF, HzS + NOTE: Subculture from Sel f and AFIIS to
(colorless with black center) sele{tiv€ media after 6-8 hours of
. Shigella - NLF, HzS- incubation
(Colorless without black
cente r)
 MICBOBIOLOGY . PLATE NEADING
+ BIOCHEMICAL TEST MEDIUM I LYSINE IRON AGAR
,/ slM {sulfide, lndole, Motility) r To determine the ability of the organism
(gram negative bacilli)to deaminate lysine,
' Sulfide: For detection of H25
. lndole: serves as media for decarboxylate lysine and produce
indole test hydrogen sulfide (H25)
. Vlotility: detects motility of , pH indicator = Bromcresol purple
. H25 indicator = Ferric Ammonium Citrate
- SLANT = Detects organism capable of
lysine deamination
(+) Red (R) = Proteus, Providencia,
Morganella
(-) Purple (K)
. BUTT = detects organism capable of lysine
decarboxylation
./ Citrate Utilization (+) Purple (K)
. To identify organisms capable (-)Yellow (A)
of using sodium citrate as the
sole carbon source and ilrr
inorganic ammonium salts as
the sole nitrogen source
. pH indicator: Bromthymol blue
,/ (r) blue = Klebsiella
( Enterobacter)
###
K/K = Escherichia coli
aerogenes
K/K H25+ = Salmonella, Arizona,
./ (-) green = E. coli
Citrobacter, Edwardsiella
,/ Triple Sugar lron
K/A = Shigella
. TSI is used to determine R/A = Proteus,Providencia,lVlorganella
whetlter a gram negative rod
ferments glucose and lactose
or sucrose and forms hydrogen
sulfide(H2S)
. The test is used primarily to
ciifferentiate members of the
Enterobacteriaceae family
from other gram negative rods
. CHO: Lactose, Sucrose,
Glucose
. pH indicator: Phenol Red to
detect acid production
. H25 Production indicator:
Ferrous Ammonium Sulfate

ffi &
a
-J

ix *A ll..& B&t
^A.X8
 MICNOBIOLOGY - PLATE BEAI'ING

BENCH RI4LE.S:
RESPIRATORY SPECIMEN:

+ Sputum
./ Gram Stain:

CONS
o Pus cells = >25/<25 per hpf
o EPithelial cells = <25/lPf

o No growth after: 24 & 48 hours *Saliva: Epithelial cells = >Zlllpf

incubation (initialland 5 days (final
report)
./ Quantity
o With growth (Positive for o Few colonies and Light growth
(not significant)
_incubation)
o Moderate to HeavY growth
-after
Positive blood c/s (18-24hrs incubation)
{significant}
,/ Significant organisms:
Significant o Staphylococcusaureus
o StrePtococcusPneumonia
o Haemophilusinfluenzae
o Klebsiellapneumoniae
Yes - mixed culture Pure colonies
o Candida albicans
Su bcu lture significa nt Bio and Sensi
organism o Enterobacteriaceae
o Moraxellacatarrhalis
,/ Significontorganisms: + Endotracheal Aspirate
o Salmonellatyphi/nontyphi ,/ Pus cells: >251<25lhpf
o Gram negative rods ,/ Quantity: Few colonies to Heavy
c Neisseria meningitidis growth
c Streptococcuspyogenes ./ Same significant organism as sputum

o Streptococcuspneumoniae + Throat Swab

o Other Streptococcus spp '/ Significantorganisms:
c Staphylococcusaurues . StreptococcusPyogenes
o Haemophilusinfluenzae . Corynebacteriumdiphtheria
o Candida spp
. Staphylococcusaureus
o Cryptococcusneoformans
. Candida albicans
o Burkholderia pseudomallei
r Moraxellacatarrhalis

Few colonies Moderate growth

) t
a'
a

Light growth
Moderately Heavy
growth
Heavy growth
 MICNOBIOLOGY - PLATE BEADING

BENCl-t RI4LE.S: o Neisseriameningitidis

o Haemophilusinfluenzae
o Streptococcus pneumoniae
Respiratory specimen (18-24hrs incubation) o Streptococcusagalactiae
o Cryptococcusneoformans
o Listeriamonocflogenes
I o Gram negative rods
Significant (?) o Staphylococcusaureus

/' Sterile Body Fluids/CSF (18-24hrs incubation)

YES - mixed culture NO - No pathogen
Subcultu re significant
organism
isolated
i
GROWTH(?)

Final report: (lD & sensi)

Pure colonies
Quantity(e.g: Light YES 1't day: NO - Reincubate primary
Bio and Sensi
growth) of orsanism. (positive _ morphology) plate and subculture from BHI

STERIIE BODY FLUIDS:

*
mixed: subculture
GROWTH(?)
Pure colonies: Bio, Sensi

+
2nd day: NO- Reincubate BHI
'/ Synovialfluid Final report: (lD & sensi)
'r Ru Ies: Positive for orsanism. t
o lf no microorganism in gram stain but
3'd day: NO - report as No
with bacterialgrowth @check cell growth after 72 hours
count: incubation
. wBc (6) URINE:
. Segmenters/Neutrophil($)
CSF: o Quantification:
-, , lul = l- col x 1000
Ru Ies . 10Ul=1colx100
o lf no microorganism but with bacterial o >100,000 col. = significant count
growth o 11,000-99,000 - check diagnosis:
./ check cell count: recurrent UTl, complicated UTl,
. wBc (6) complete a ntibiotic treatment
r SegmenterslNeutrophil($) o <10,000 - not significant EXCEPT
,/ csF Glucose:
S Yeast/Candida
 MICROBIOLOGY . PLATE BEADING

SCREENING TEST for Methicillin-Resistant

Staphylococcus aureus

Principle:
Tests for mecA or for the protein
expressed by mecA, the penicillin-binding
protein 2a (PBP 2a, also called PBP 2') are
the most accurate methods for prediction of
resistance to oxacillin and can be used to
confirm results for isolates of Staphylococci
from serious infection. lsolates of
Staphylococci that carry the mecA gene, or
that produce PBF2a (the mecA gene product)
should be reported as oxacillin resistant. This
can be detected by testing cefoxitin disk 30 ug
and is used as surrogate for mecA-mediated
oxacillin resistance.

Procedure:
1. Following the standard disk diffusion, place
cefoxitin disk onto plain [/ueller Hinton agar.
2. lncubate for 16-18 hours.
3 Read susceptibllity results (read with
transmitted light) and interpret according to
the lates CLSI document.
 MICNOBIOLOGY . PLATE BEADING

REPORTING OF RESULTS SCREENING TEST FOR ESBL USING

DOUBLE DISK DIFFUSION METHOD
SUSCEPTIBLE implies that isolates are
(s) inhibited by the usually Principle:
achievable concentrations Extended Spectru m Beta-Lactal??ases
of antimicrobial agent (ESBL)are enzymes that confer resistance to
when the dosage extended-spectrum cephalosporins and
recommended to treat the monobactams but do not affect cephamycins or
site of infection is used carbapenems. ESBL production can be detected
by double disk test using a disk containing
SUSCEPTIBLE. implies that susceptibility clavulanic acid (Ah/C) and placing it between
DOSE of an isolate is dependent cefotaxime and aztreonam disk. An elliptical
DEPENDENT on the dosing regimen that clearing or distortion of inhibition in synergistic
(sDD) is used in the patient. fashion (keyhole) between the 2 disks indicates
the inhibition of B-lactamase by clavulanic acid.
INTERMEDIATE implies clinical efficacy in
(t) body sites where the drugs Procedure:
are physiologically 1. Following the general procedure for
concentrated or when a antimicrobial susceptibility testing, place
higher than normal dosage Aztreonam, Amoxicillin/Clavulanic Acid,
can be used. Cefotaxime and lmipenem
2. lncubate plates for 18-24 hours.
NON- used for isolates for which 3. Examine the plate for ESB-production
SUSCEPTIBLE only a susceptible
(NS) interpretative criterion has lnterpretation:
been designated because . Positive. A clear extension (keyhole or
of the absence or rare ellipse) of the edge of cefotaxime or
occu rrence of resistant aztreonam inhibition
strain. zone toward the disk
containing clavulanate
RESTSTANT (R) implies that isolates are . Negative.' No keyholes or ellipse
not inhibited by the between Aztreonam,
usually achievable Amoxicillin/Clavu lanic Acid and
concentration of agent with Cefotaxime
normal dosage schedules,
and/or that demonstrate
[tIlCs or zone diameters
that fall in the range where
specific microobial
resistanc mechanism.
 MICROBIOLOGY - PLATE BEADING

lsolation of various Hoemophilus species on the basis of Srowth SELECTING ANTIMICROBIAL AGENTS
chrracteristics: FOR TESTING AND REPORTING
Speci€s X C0r Hemolysis on horse
blood aear TIER I routine primary testing
panel as well as for
.,i routine reporting of
+ results

+
TIER II includes antimicrobial
agents that may warrant
Note: H. influenzae is xylose positive where as H. **tsyptius ne*ative.
primary testing but they
X€y: X -factor (H€minf- lt t5 requlred for synthesis ol resplratory enzyme and .lso
maybe reported
called heat stable growth prmoting subttancs Pr6ent in Red Blood Cells.
selectively.
v-factor {nkotinamide-bdenine dinucleotideftA0) - k ls ued as an electron carrle
alss lnown a a heat labile Vltsmin li&e tubstEnces P.esent ln Red Bl@d Cellr'
GROUP C includes alternative or
supplemental
antimicrobial agents that
may require testing in
those institution that
harbor endemic or
epidemic strains resistant
to several of the primary
drugs; for treatment of
patients allergic to
primary drugs; for
reporting to infection
control as an
epidemilogical aide

GROUP lnv includes antimicrobial

('investigational") agents that are
investigational for
organism group and have
not yet been approved by
FDA

GROUP U ("urine") includes antimicrobial

agents that are used only
or primarily for treating
urinary tract infection.

GROUP O antimicrobial agents that

("other") have clinical indication for
organism group, but are
generally not candidates
for routine testing and
reportinq.
 MICROBIOLOGT - PL.ATE READING

GTR AND ABSCESS (18-24hrs incubation)

J

GROWTH(?)
)
{
a
YES .
1't day: NO - Reincubate
(quantity morphology) primary plate and subculture
-
from THIO

Not significant growth. v

Report as: Work up significant
orga nisnr
GROWTH(?)
NO PATHOGEN ISOTAIED
{ mixed: subculture
Pure colonies: Bio, Sensi
I
"1
I
- Reincubate THIO
I
Final report: (lD & t
sensi) report as No growth
3'd day: N0 -
Quantity (e.g: Light after 3 days of incubation
erowth) of orsanism
 MICROBIOLOGY - PLATE BEADING
o > 3 microorganisms - report as
Conta mi no nts isolated. Re peat

collection suggested.

SItENITE
o Escherichia coli
tvlACISM
AC F

o Other Gram negative rods

o Enterococci
o Staphylococcussaprophyticus :. ,: r,:rr:'-. :..11 ','l\ iii ,:' j ,r :,ir.i : l

o Pseudomonas and other non-

fermenters TC I}!
55A
o Other Staphylococci .
1 1

ir:_r!t
ri i.rr
Ii

o Candida albicans

STOOL/RECTAL SWAB
. f
:!._( lll:ic)it!,i ri : 'r:. i i : - \

o lD first before sensitivity testing

o Mac - Non- Lactose Fermenter (Bio/lD WOUND SWABfi ISSUE/ABSCESS/ASPIRATE
first)
o Salmonella and Shigella Agar ,/ Quantify growth as: Few colonies, Light
,/ NLF
growth, Moderate growth, Modera
,/ NLF with black center (salmonella ,/ tely heavy growth and Heavy growth
tvphi)
/ Significant organisms:
o TCBS
o Staphylococcusaureus
/ Large flat yellow * Vibrio cholera
{ Large flat green
o Streptococcuspyogenes
- Vibrio
o Enterobacterales
parahaemolyticus
o Pseudomonas and other non-
fermenters
{ Salmonella
{ shigella
o Other Streptococcus species
/ vibrio
o Candida

r' Plesiomonas
/ Aeromonas GTR - PENILE AND VAGINAL SWAB
./ Positive for pathogenic organism
x Quantify growth as: Few colonies, Light
No important enteropathogen
growth, Moderate growth, Moderately
isolated.
heavy growth and Heavy growth
Significant organisms (Vaginal swab)
o Neisseria gonorrhoeae
o Candida albicans
o Streptococcusagalactiae
Significant organisms (Penile swab- STI)
o Neisseriagonorrhoeae