BACTERIOLOGY

TRAINING
PREPARED BY: MA. RIOLYNNE C. DELA CRUZ
MICROORGANISM
MICROORGANISM

◦ COCCI
◦ BACILLI
◦ DIPLOCOCCI
◦ COCCOBACILLI
◦ DIPHTHEROIDS

◦ YEAST
◦ HYPHAE
COCCI IN
CLUSTERS
- Staphylococcus spp
- Micrococcus spp.
COCCI IN
CHAINS
- Streptococcus
viridans
- Streptococcus
agalactiae
- Streptococcus
pyogenes
- Group D
Streptococcus
COCCI IN
PAIRS
-Streptococcus
pneumoniae
DIPLOCOCCI
- Neisseria
BACILLI
- Lactobacillus
- Listeria
- Corynebacterium
- Enterobacteriacea
(Escherichia, Klebsiella,
Enterobacter,
Citrobacter. Etc)
COCCOBACILLI
- Haemophilus
influenzae
- Acinetobacter
DIPHTHEROIDS
- Corynebacterium
diptheriae
YEAST &
HYPHAE
- Candida albicans
- Candida spp.
GRAM STAINING
 GRAM
STAINING
PRINCIPLE
 GRAM STAINING

V- CRYSTAL VIOLET

I- GRAM’S IODINE

A- ACETONE ALCOHOL

S- SAFRANIN
SMC LAB SOP:
DIRECT STAINING COLONY STAINING

◦ V- 1 MINUTE ◦ V- 1 MINUTE
◦ I- 1 MINUTE ◦ I- 1 MINUTE
◦ A- 30 SECONDS ◦ A- 10 SECONDS
◦ S- 10 SECONDS ◦ S- LESS THAN 10
SECONDS
 GRAM STAINING
GRAM POSITIVE GRAM NEGATIVE
All COCCI are GRAM POSITIVE
Except:
◦ Neisseria MNEMONICS:

◦ Branhamella
◦ Moraxella
No
◦ Veilonella Boyfriend
Muna (para)
V…
All BACILLI are GRAM NEGATIVE
Except:
◦ Bacillus MNEMONICS:
◦ Lactobacillus
◦ Listeria
◦ Actinomyces
BL2AC2ME
◦ Clostridium
◦ Corynebacterium
N
◦ Mycobacterium
◦ Erysepelothrix
◦ Nocardia
LACTOSE FERMENTER
VS.
NON-LACTOSE
FERMENTER
LACTOSE FERMENTER NON-LACTOSE FERMENTER
-pink colonies - colorless colonies
LACTOSE FERMENTERS
◦ Citrobacter **
◦ Enterobacter
◦ Escherichia
◦ Klebsiella
MNEMONICS:

CEEK
NON-LACTOSE FERMENTERS
◦ Shigella
◦ Yersinia
◦ Proteus
◦ Pseudomonas
◦ Salmonella MNEMONICS:

ShYPPS
LATE-LACTOSE FERMENTERS
◦ Citrobacter ** MNEMONICS:
◦ Arizonae
◦ Providencia CAPS
◦ Serratia
LF OR NLF?
HEMOLYSIS
 COLONY
MORPHOLOGY
◦ SIZE: PINPOINT SMALL MEDIUM LARGE

◦ COLOR:

◦ ELEVATION:

◦ APPEARANCE
◦ Texture: DRY SMOOTH VISCID MUCOID

◦ Form:

◦ Margination:

◦ HEMOLYSIS PATTERN: ALPHA BETA GAMMA

 BASIC
BIOCHEMICAL
TESTS
BASIC MANUAL WORK-UPS
◦ TSIA ◦ CATALASE
◦ LIA ◦ COAGULASE
◦ SIM ◦ TAXO-A
◦ CITRATE ◦ TAXO-P
◦ VP ◦ BILE SOLUBILITY TEST
◦ UREASE ◦ BILE ESCULIN HYDROLYSIS
◦ OXIDASE ◦ 6.5 % NACL
◦ SUGAR FERMENTATION TESTS
TSIA
◦ - TRIPLE SUGAR IRON AGAR
◦ Used to differentiate among the different groups of
Enterobacteriaceae
◦ Detects three primary characteristics of a bacterium:
◦ 1. The ability to ferment sugars.
◦ 2. The ability to produce gas from the fermentation of sugars,
◦ 3. production of large amounts of hydrogen sulfide.

How to Perform Test: Stab through the center of the butt up to the bottom
of the tube then draw out and from the lower portion of the slant make a
vertical streak then fishtail over slant.
TSIA
◦ INTERPRETATION OF RESULTS:
TSIA
◦ INTERPRETATION OF RESULTS:
TSIA
TSIA
RESULTS: (From left to right)
◦ Morganella morganii (K/A,
atypically not producing
gas),
◦ Pseudomonas
aeruginosa(K/NC),
◦ Uninoculated control,
◦ Proteus mirabilis(K/A,H2S),
◦ Escherichia coli (A/A,G)
TSIA MNEMONICS:

◦ A/A
CEEK
◦ Citrobacter H2S (+)
◦ Escherichia mostly (-) E.coli: Indole (+)
◦ Enterobacter mostly (+)
◦ Klebsiella non-motile
TSIA MNEMONICS:

◦ K/A SS PMP YE
◦ Salmonella H2S (+)
◦ Shigella non-motile
◦ Proteus H2S (+)
◦ Providencia Citrate (+)
◦ Morganella Urease (+)
◦ Edwardsiella mostly (-)
◦ Yersinia “safety pin” on CIN
TSIA MNEMONICS:

PABS
◦ K/K
◦ Pseudomonas oxidase (+)
◦ Acinetobacter hard to grow/ do not grow on MAC
◦ Burkholderia
◦ Stenotrophomonas
LIA
◦ - LYSINE IRON AGAR
◦ A agar is used to determine whether a Gram negative rod
decarboxylates or deaminates Lysine
◦ Lysine deamination is an aerobic process which occurs on the slant of
the media.
◦ Lysine decarboxylation is an anaerobic process which occurs in the
butt of the media. •
How to Perform Test: double stab and streak.
LIA
◦ LYSINE DEAMINATION (SLANT)
◦ (+) RED
◦ (-) PURPLE
◦ LYSINE DECARBOXYLATION (BUTT)
◦ (+) PURPLE
◦ (-) YELLOW
LIA
PMP GROUP
R/Y (+/-)
Proteus
Morganella
Providencia
 SIM
◦ SULFIDE INDOLE MOTILITY
◦ Sulfide production (Presence of thiosulfate reductase
enzyme)
◦ Indole production (Presence of tryptophanase enzyme)
◦ Motility Detection

How to Perform Test: stab once through the center of the agar to a
depth of 1/3 or 1/2 of the medium.
SIM
 SIM
◦ (A) uninoculated tube
◦ (B) contains the nonmotile and
indole negative bacterium
◦ (C) contains the motile and indole-
positive bacterium
◦ (D) contains the motile, indole-
negative, and H2S-producing
bacterium
 CITRATE UTILIZATION TEST
◦ This test is used to help differentiate species of the
family Enterobacteriaceae. It is selective for bacteria
that has the ability to consume citrate as its sole source
of carbon and ammonium as sole nitrogen source.

How to Perform Test: Inoculate slant with inoculating

loop.

INTERPRETATION:
(+) BLUE
(-) GREEN (NO COLOR CHANGE)
UREA HYDROLYSIS TEST (UREASE)
◦ To determine if the microorganism that possesses the
enzyme urease that can hydrolyze urea, releasing ammonia
and producing a pink red color change in the medium •

How to Perform Test: Make a vertical streak then

fishtail over slant

INTERPRETATION:
(+) SALMON PINK
(-) NO COLOR CHANGE
VOGUES PROSKAUER (VP)
◦ Determines the ability of the organism to produce acetoin or
acetyl-methyl carbinol as the chief end product of glucose
fermentation.

How to Perform Test: Inoculate MRVP broth. After 16-18

hours of incubation, add 0.6 ml (6 drops) of 5% α-
naphthol, Followed by the addition of 0.2 ml (2 drops) of
40% KOH.

INTERPRETATION:
(+) DEEP ROSE COLOR AFTER 15 MINUTES
(-) NO COLOR CHANGE
OXIDASE TEST
◦ To determine the presence of bacterial cytochrome oxidase

INTERPRETATION:
(+) PURPLE TO BLUE
(-) NO COLOR CHANGE

Enterobacteriacea are Oxidase

NEGATIVE
CASE STUDY 1:
CASE STUDY 1 (ANSWER)
◦ TSIA A/A

◦Escherichia coli
◦ LIA -/+
◦ SIM - ++
◦ CITRATE V
◦ VP V
◦ UREASE V
◦ OXIDASE -
CASE STUDY 2:
CASE STUDY 2 (ANSWER)
◦ TSIA K/A

◦Proteus mirabilis
◦ LIA +/-
◦ SIM +v+
◦ CITRATE V
◦ VP V
◦ UREASE V
◦ OXIDASE -
CATALASE
◦ Reagent: 3 % H2O2 (Hydrogen Peroxide)

POSITIVE: “Effervescence” or bubble

formation

*** Superoxol Test:

◦ Uses 30% H2O2
COAGULASE (SLIDE)
◦ CLUMPING FACTOR
◦ Detects “bound” coagulase

POSITIVE: “Clumping”
COAGULASE (TUBE)
◦ Detects “FREE” coagulase
◦ Reagent: Rabbit Plasma OR Citrated Human
Plasma
◦ 2HRS, 4HRS, 24 HRS

POSITIVE: “Clot-formation” or Coagulum

OPTOCHIN TEST (TAXO P)
◦ Optochin Disc or Taxo “P” Disc

◦ 1. Select an isolated colony

◦ 2. Streak onto half of plate
◦ 3. Drop disk on streaked area
◦ 4. Incubate in CO2 , 35-37⁰C
OPTOCHIN TEST (TAXO P)
BILE SOLUBILITY TEST
BILE
SOLUBILITY
TEST
BILE ESCULIN TEST
6.5 % NACL
BACITRACIN TEST (TAXO A)
◦ Uses: Taxo A Disc
◦ Differentiation of
Streptococcus
pyogenes and
Streptococcus
agalactiae
CAMP TEST
CAMP TEST
HIPPURATE TEST
◦ POSITIVE: BLUE COLOR
◦ NEGATIVE: COLORLESS
PYR TEST
SUGAR FERMENTATION TEST
◦ (-) reaction =
Orange or no
change in color
◦ (+) reaction =
Yellow
STREAKING
TECHNIQUE
Streaking Technique
 MDRO
(MULTI-DRUG RESISTANT ORGANISMS)
CEFOXITIN SCREEN TEST
◦ Detects mecA Gene
◦ Detect OXACILLIN Resistance but uses
CEFOXITIN Disk
◦ Used to detect MRSA

POSITIVE RESULT:
◦ ZONE OF INHIBIITION:
◦ </= 21 mm (RESISTANT) for S.aureus
◦ </= 24 mm (RESISTANT) for CoNS
CEFOXITIN SCREEN TEST
REPORTING:
◦ Cefoxitin Screen POSITIVE (Cefoxitin
Disk)
◦ mecA Gene POSITIVE (Vitek)

Release as:
◦ Methicillin Resistant Staphylococcus
aureus (MRSA)
◦ Methicillin Resistant Coagulase-
Negative Staphylococcus spp (MR-
CoNS)
ICR (INDUCIBLE CLINDAMYCIN
RESISTANCE
◦ Detects MLSB gene and erm
gene
◦ Used to identify Macrolide
Resistant Staphylococcus
species
◦ Can be detected by disk
approximation test (D-zone Test)
using Clindamycin disc against
Erythromycin disc.
ICR (INDUCIBLE CLINDAMYCIN
RESISTANCE
◦ ICR (+) = Flattening of
clindamycin zone adjacent to
erythromycin disk; “D-ZONE”

◦ ICR (-) = No flattening of

clindamycin zone adjacent to
erythromycin disk
ICR
 ICR
(INDUCIBLE
CLINDAMYCIN
RESISTANCE
 ESBL SCREENING
◦ Extended Spectrum Beta-lactamase (ESBL)
◦ Produced by Enterobacteriaceae

◦ Antibiotics Involved:
◦ Aztreonam (ATM)
◦ Clavulanate like Amoxicillin-Clavulanic Acid
(AMC)
◦ Cefotaxime (CTX)
ESBL SCREENING
◦ POSITIVE RESULT:

◦ “KEYHOLE” or
“ELLIPSE” at the edge
of CTX and/or ATM
ESBL
CONFIRMATORY
◦ POSITIVE RESULT:

◦ “PHANTOM”
AMPC
◦ AmpC Beta-lactamase / Class C / Group 1
Beta-lactamase
◦ Produced by some Gram-negative bacteria

◦ Antibiotics involved:
◦ Imipenem (IPM)
◦ Clavulanate (AMC)
◦ Cefoxitin (FOX)
◦ Aztreonam (ATM)
◦ Cephalosporins (Cefotaxime CTX, Ceftazidime
CAZ, Etc.)
AMPC
◦ POSITIVE RESULT:

◦ “FLATTENING” or
“DISTORTION” due to
Antagonistic Effect

◦ May appear as “D-shaped”

or “Guitar-pick”
Other MDROs:
◦ Carbapenemase (mCIM/eCIM)
◦ Metallo-Beta-lactamase
◦ Serine-Beta-lactamase
 THE END
THANK YOU FOR LISTENING!!! 