Catalase test

 Test used to differentiate between staphylococci and

streptococci
 Used to know weather the bacteria has the catalase
enzyme or not
 The catalase enzyme breakdown the hydrogen
peroxide into water and oxygen
 Positive results shown as air pupples in the tube
 Positive indicates staph
 Negative indicates strepto
 Oxidase test

Used in differentiation between campylobacter,vibrio,
pseudomonas from other gram negative bacilli.
 Used to know if the bacteria produces oxidase enzyme or not.
 Oxidation of phnylenediamine will change it to purple color if
it is positive in seconds.
 The name of the reagent is tetramethyl-p-phnylenediamine
dihydrochloride.
 Bile solubility test
 Used in differentiation between s.pneumoniae and viridians.
 The principle goes upon the ability of the bile salts sodium
deoxycholate for dissolving of the organism and remove the
turbidity which occurs from emulsify the organism in
physiological saline.
 Positive results indicates pneumoniae

 CAMP test
 Used in identification of S.agglactiae

 Camp factor is an extracellular diffusible protein

 Interacts with the beta lycine produced by S.aurues

 The both organisms are cultured in one plate across each other

 The interaction appears as arrow head

 Citrate utilization test
 Used to determine the ability of the organism to grow
when the citrate is the only source of carbon and
ammonia is the only source of nitrogen.
 Culture the organism in a media contain sodium
citrate and ammonium salts with bromo thymol blue
as indicator
 Changing of the color from light green to blue
indicates that the organism can grow and utilize
citrate
 Media used maybe koser citrate media or simmon
citrate agar
 Coagulase test
 Used in differentiation between s.aurues and other staph which
produce coagulase enzyme which is two types
 Free coagulase which convert fibrinogen to fibrin by
activation of coagulase reacting factor (tube method)
 Bound coagulase which convert fibrinogen to fibrin directly
(slide method)
 Agglutination of bacteria indicates it is s.aurues and the test is
positive
 Indole test
 Used to show the ability of the bacteria to break the
amino acid tryptophan.
 When tryptophan is broken three substances are
produced one of the is indole
 The organism is cultured in pepton water containing
tryptophan and after incubation Kovac reagent is
added to detect the presence of indole.
 Kovac reagent contains 4-p-
dimethylaminobenzaldehyde
 If the test is positive the compound will react with the
indole producing red color
 Litmus milk test
 Used in identification of fecealis

 Incubation of the organism in tube containing litmus milk the

organism will reduce the color from mauve to white or pale
yellow
 Nitrate reduction test
 Used in differentiation between enterobacteriaceae
and other gram negative bacilli
 To know If the organism have the enzyme nitrate
reductase which reduce nitrate to nitrite or not.
 Incubate the organism in a broth containing nitrate
 Add one drop of sulphanilic acid and alpha-
naphthylamine
 If nitrite is present the acid is diazotized and forms a
pink-red component with the alpha-naphthylamine
 If no red color is present that means either the test is negative
or the organism reduce nitrate to other substances(nitrogen gas
or ammonia)
 So we add Zink dust to make sure
 Zink dust will convert any nitrate to nitrite and will give red
color
 So if you found any red color after adding Zink dust that
means the test is negative because the organism did not reduce
the nitrate
 But is there was no red color the test is positive but the
organism reduced nitrate to other substances not nitrite
 Deoxyribonuclease test
 Used in identification of s.aurues
 We culture the organism in media contain DNA
 If the bacteria has the DNAs enzyme will hydrolyze the DNA
around it
 Then we add weak HCL on the media it will precipitate the un
hydrolyzed DNA
 If the organism is s.aurues clear zone will be around it
 That indicates it hydrolyzed the DNA by the enzyme
 Oxidation fermentation test
 Used to differentiate organisms that oxidize CHO from those
which ferments it.
 The organism is incubated in two tubes of peptone agar
contains glucose with bromo-thymol blue as indicator
 Seal one tube with layer of liquid paraffin wax to exclude
oxygen
 Incubate and wait for results
 Fermentative organism will utilize CHO in both tubes and
change color from green to yellow
 Oxidative organism will only utilize CHO in the open tube in
the presence of oxygen
 Phenylalanine test
 Used to assist in identification of enterobacteria which have
the ability to breakdown phenylalanine to phenylpyruvic acid
by oxidative deaminatoin

 The product phenylpyruvic acid is detected by adding ferric

chloride

 Appearance of green color the test is positive

 Urease test
 Testing of urease enzyme activity is Used in differentiation
between enterobacteria species.
 Incubate the organism in media containing urea with indicator
phenol red
 Urease enzyme will break urea to ammonia and carbon
dioxide which lead to give an alkaline media that changes the
color of the indicator to pink
 Motility indole urea is used
 Methyl red and voges proskaur
 In utilization of CHO some organisms produces a
large amount of acids which can be detected by MR
 Other organisms can form
acetylmethylcarbinol(acetoin) which can be detected
by V-P.
 In MR the organism is cultured in glucose phosphate
broth then add a drop from MR if red color appeared
the test is positive
 IN VP we add sodium hydroxide and creatine
powder, the acetoin produced is oxidized to diacetyl
which forms a pink compound with creatine
 KIA media
 This media is composed of:-
1. Butt and slant
2. Glucose 1gm
3. Lactose 10gm
4. Phenol red as indicator
5. Sodium thiosulfate and ferric citrate
 Some organisms ferment only glucose so yellow
color will appear then the organism will attack the
amino acids in the slant
 In this case yellow butt and red slant
 Some organisms can ferment both glucose and lactose in this
case all the media is yellow


 Some organisms which ferment only glucose produces

hydrogen sulfide

 Hydrogen sulfide produce black color in the meduim

 Some organisms produce gas which can be seen as cracks in

the media or lead to elevation of the media
 According to the reactions seen the organism can be classified
into:-

 Lactose fermented or not

 Hydrogen sulfide producer or not

 Gas producer or not

 Result
Example s
Result
Reaction on TSI
H2S Slant color Butt color
LNF A/Alk/- Negative
Red Yellow
e.g. Shigella (Glucose fermented)
LNF A/Alk/+ Positive
e.g. Salmonella & (Glucose fermented with H2S) black in butt Red Yellow
Proteus
LF
A/A/-
e.g. E. coli, Klebsiella, Negative Yellow Yellow
(three sugars are fermented)
Enterobacter
Non fermenter e.g. Alk/Alk/-
Red Red
Pseudomonas (No action on sugars) Negative
 Macconkey agar
 Very useful media


 Works as:-

 differential lactose fermented or not

 Selective media for gram positive bacteria(bile salts)

 Pyogens can not grow in this media

 Differentiation between LF & NLF by Growth on MacConkey agar

 MacConkey agar is selective & differential medium for Enterobacteriaceae

MacConkey Agar
Contains

Bile salts Crystal violet Lactose Neutral red

pH indicator
Inhibit growth of G+ve bacteria Cause of differential Acidic: Pink

Cause of selectivity
Lactose feremnters Lactose non feremnters
Pink colonies colorless colonies
 Growth of Enterobacteriaceae on
MacConkey agar

Colorless colonies Pink colonies

Uninoculated plate Lactose non feremters Lactose feremters

Salmonella, Shigella, E. coli, Citrobacter
Proteus Klebsiella, Enterobacter
Reaction on Salmonella Shigella (SS) agar
 SS agar is a selective & differential medium used for isolation
of Salmonella and Shigella

 The selective agents are bile salts, and brilliant green dye,
which inhibit gram-positive organisms

 The medium contains only lactose as a differential agent and

thus differentiates on the basis of lactose fermentation

 The formation of acid on fermentation of lactose causes the

neutral red indicator to make pink colonies

 Non lactose fermenting organisms are colorless on the

medium
 SS agar contains sodium thiosulfate and ferric ammonium
citrate allows the differentiation of organisms that produce
H2S
 Lactose fermenters, such as E. coli, have colonies which

are pink
 Shigella appears transparent or amber

 Salmonella appears transparent or amber with black centers

due to H2S production

Lactose fermenter Neutral red

Lactose Acid Pink colonies

H2S + Ferric ammonium citrate Ferrous sulfide

Black precipitate
 Growth of Enterobacteriaceae on SS agar

A. Klebsiella pneumoniae
Both are lactose fermenters
B. Escherichia coli
C: Salmonella sp. Both Salmonella sp. & Proteus product H2S
D: Proteus mirabilis
E: Ps. aeruginosa Pseudomonas colonies are nearly colorless
.
 Deoxycholate citrate agar
 Selective and differential media used to isolate
salmonella and shigella

 May be used to isolate yersinia if we increased the

percentage of the bile salts

 Composed from lactose soduim citrate soduim

thiosulfate
 ferric citratE
 The indicator is neutral red which is pink in acidity
Dorset egg and leoffler serum media
 These two medias are used as:-

1. Enriched media used to culture

corynebacterium diohtheriae

2. Used to detect volutin granules

 Modified tinsdale media(MTM)
 Selective and differential media used to isolate
corynebacteruim.D

 Too expensive commercially

 Glucose phosphate pepton water
 Fluid media used in:-

1. Methyl red test

2. Vogues proskauer test

 Lactose egg yolk milk agar
 Differential media used for unaerobes specialy
clostriduim species
 Clostriduim are differentiated by:-
1. lactose fermentation
2. libase activity
3. lecithinase production
 The media can be selective for clostriduim by
adding neomycin sulphate
 Manitol salt agar
 Differential and selective media

 Used to isolate and differentiate S.aurues from

other staph bacteria

 Contain lactose for differentiation and phenol

red as indicator
 Xylose lysine deoxycholate agar
 Used to isolate salmonella and shigella
 Depends on:-
 Xylose fermentation
 ylsine decarboxylation
 Hydrogen sulfide production
 Contains xylose-lysine-sucrose-lactose
 The indicator is phenol red
 Citrine lactose electrolytes deficient
media(CLED)
 Valuable non inhibitory media

 Prevent swarming of protues due to electrolyte deficient

 Bromo thymol blue is the indicator

 Lactose fermenting produce yellow colonies

 Non lactose produce blue colonies

 Thiosulphate citrate bile salt:-
 Selective media used to isolate vibrio cholerae

 Tellurite blood agar

 Selective media used to isolate corynebacteruim.D

 Selenite broth:-
 Used as enrichment media for the isolation of
salmonella species

 Modified new york city

 Selective media for isolation of nisseria gonorrhoeae
 Bacteria and gram stain
 Bacteria according to gram stain is devided to:-
1. Gram positive cocci
2. Gram negative cocci(we only have nisseria)
3. Gram positive bacilli
4. Gram negative bacilli
5. Gram negative small rods or coccobacilli
G-ve bacilli G+ve bacilli G-ve coccob G+ve cocci
E.coli bacillus yersinia s.aurues

Klebsiella Coryne.B brucella s.epidermidis

Salmonella clostridium H.influenzae s.saprophyticus

shigella Listera bordetella st.pyogenes

protues St.agalactiae

psuedomonas St.fecealis

vibrio St.viridans

campylobacter St.pneumoniae

Bacteriod
 In this groub we have 8 organisms devided into two
groups
 Staphylococcus which include:-
1. Aurues
2. Epidermidis
3. Saprophyticus
 Streptococcus which include
1. Pyogens
2. Aglactiae
3. Pnuemoniae
4. Viridans
5. Fecealis
 According to catalase test
 Catalase positive  Catalase negative

 sataphylococcus  streptococcus
 Differentiation between first group members

epidirmidis saprophyticus Aurues test

VE- VE- VE+ Coagulase

ve weak+ VE- VE+ DNAs

VE- VE+ VE+ Manito salt

agar.F

sensitive resistance sensitive Novobiocin

sensitivity
 Identification of streptococci
 According to the type of haemolysis on
blood agar devided into:-
 Beta haemolysis(pyogens and agalactiae)

 Alpha haemolysis(pnuemoniae,viridans)

 Non hemolytic ( fecealis)

Strepto pyogens
 Catalase negative
 Beta haemolysis
 Crystal violet is the selective media
 Does not grow on macconkey
 Sensitive to bacitracin
 PYR positive
 Strepto aglactiae
 Catalase negative
 Beta haemolysis
 Kanomycin blood agar is the selective media
 Can grow on macconkey
 CAMP test is positive
 Hipurate hydrolysis positive
 Pnuemoniae and viridans
 Catalase negative
 Alpha haemolysis
 Tests used for differentiation between them

viridans pnuemoniea Test

Resistance Sensitive Opticin

sensitivity
VE- VE+ Bile solubility
 Strepto fecaelis
 Catalase negative

 Non haemolytic

 Can grow in 6.5 NACL

 Ferment lactose on macconkey agar

 Hydrolize asculin

 Reduce litmus milk

 Sensitive to ampcilin
CAMP L.M OPT BAC catalase species
BILE.S
VE- VE- VE- VE+ VE- Pyogens

VE+ VE- VE- VE- VE- Aglactiae

VE- VE- VE+ VE- VE- Pnuemoniae

VE- VE- VE- VE- VE- Viridans

VE- VE+ VE- VE- VE- Fecaelis

 According to oxidase test
 Oxidase positive  Oxidase negative

1. Vibrio
1. E.coli
2. Psuedomonas
2. Klebsiella
3. Salmonella
3. campylobacter 4. Shigella
5. Protues
 Oxidase negative group
 According to lactose fermenting on macconkey or
KIA we can devide the into

1. Lactose fermenter which include E.COLI and

KLEBSIELLA

2. Non lactose fermented which include

salmonella,shigellaand protues
Differentiation between E.coli and klebsillae

klebsiella E.coli Test

VE+ VE- Citrate

VE+ VE- Urease

VE- VE+ Motility

VE- VE+ Indole

 Differentiation between salmonella shigella and
protues.
1. In KIA according to hydrogen sulfide formation
shigella does not form hydrogen sulfide
2. We can differentiate between protues and
salmonella by citrate and urease tests
3. Salmonella typhi do not produce gas
4. Salmonella paratyphi do not produce hydrogen
sulfide,so we differentiate it from shigella and
salmonella typhi by the gas production.
5. We differentiate between the protues speceis by
indole test
Gas H2s Urea Ind CIT Suc speceis

ve- ve- ve- ve- ve- ve- shigella

ve- ve+ ve- ve- ve- ve- s.Typhi

ve+ ve- ve- ve- ve- ve- s.Paratyphi

ve+ ve+ ve+ ve+ ve+ ve+ P.Vulgaris

ve+ ve+ ve+ ve- ve+ ve+ P.mirabilis

 Only salmonella paratyphi.A do not produce hydrogen sulfide
 Diffrentiation between the two protues type only by indole test

 Protues vulgaris +ve indole

 Protues mirabilis -ve indole
 E.Coli
 On blood agar:-
 1-4mm colonies may appear mucoid and some are hemolytic

 On macconkey agar:-
 Lactose fermenting colonies

 Most strains do not grow on SS,XLD,DCA

 Biochemical reactions:-
1. Lactose positive
2. Manitol positive
3. Glucose positive
4. Citrate negative
5. Motility positive
6. Indole positive
7. Urease negative
8. Slope of KIA yellow
9. Butt of KIA yellow
10. H2S production negative
11. Gas production positive
 klebsiellae
 On blood agar
 Large mucoid colonies

 On macconkey agar
 Lactose fermenting colonies
 Biochemical reactions:-
1. Lactose positive
2. Manitol positive
3. Glucose positive
4. Citrate positive
5. Motility negative
6. Indole negative
7. Urease positive
8. Slope of KIA yellow
9. Butt of KIA yellow
10. H2S production negative
11. Gas production positive
 Shigella
 Xylose lysine deoxycholate(XLD):-
 Forms red colonies because it does not ferment xylose, lactose
and sucrose
 Salmonella shigella media
 Shigella appears transparent
 Deoxycholate citrate agar
 Non lactose fermenting colonies
 Cary blair is used as transport medi for shigella
 On macconkey non lactose fermenting colonies
 Biochemical reactions:-
1. Lactose negative
2. Manitol sometimes
3. Glucose positive
4. Citrate negative
5. Motility negative
6. Indole sometimes
7. Urease negative
8. Slope of KIA red
9. Butt of KIA yellow
10. H2S production negative
11. Gas production negative
 Salmonella
 XLD:-
 Salmonella produce red colonies inspite of their fermentation
to xylose with acid production.?(WHY?)
 Salmonella shigela media:-
 Appear transperant with black centers dye to hydrogen sulfide
 DCA and Macconkey
 Produce non lactose fermenter
 Biochemical reactions salmonella typhi:-
1. Lactose negative
2. Manitol positive
3. Glucose positive
4. Citrate negative
5. Motility positive
6. Indole negative
7. Urease negative
8. Slope of KIA red
9. Butt of KIA yellow
10. H2S production positive
11. Gas production negative
 Biochemical reactions S.paratyphi A:-
1. Lactose negative
2. Manitol positive
3. Glucose positive
4. Citrate negative
5. Motility positive
6. Indole negative
7. Urease negative
8. Slope of KIA red
9. Butt of KIA yellow
10. H2S production negative
11. Gas production positive
 Protues
 XLD and SS
 Non lactose fermenting colonies with black centers due to
hydrogen sulfide production

 DCA and macconkey

 Non lactose fermenting

 Blood agar
 Shows swarming
 Biochemical reactions protues vulgaris:-
1. Lactose negative
2. Manitol negative
3. Glucose positive
4. Citrate sometimes
5. Motility positive
6. Indole positive
7. Urease positive
8. Slope of KIA red
9. Butt of KIA yellow
10. H2S production positive
11. Gas production positive
 Biochemical reactions protues vulgaris:-
1. Lactose negative
2. Manitol negative
3. Glucose positive
4. Citrate sometimes
5. Motility positive
6. Indole negative
7. Urease positive
8. Slope of KIA red
9. Butt of KIA yellow
10. H2S production positive
11. Gas production positive
 This group contains:-

1. Campylobacter

2. Pseudomonas

3. Vibrio
 Differentiation between vibrio and
psuedomonas:-

 vibrio is indole positive but psuedomonas is

negative

 By oxidation fermentation test

 Vibrio ferment glucose but psuedomonas oxidize it

 KIA
1. In vibrio yellow butt and red slant
2. In psuedomonas red slant and butt
 Biochemical reactions of vibrio cholerea :-
1. Lactose negative
2. Manitol positive
3. Glucose positive
4. Citrate sometimes
5. Motility positive
6. Indole positive
7. Urease negative
8. Slope of KIA red
9. Butt of KIA yellow
10. H2S production negative
11. Gas production negative

Biochemical reactions of vibrio parahaemolyticus:-
1. Lactose negative
2. Manitol positive
3. Glucose positive
4. Citrate sometimes
5. Motility positive
6. Indole positive
7. Urease negative
8. Slope of KIA red
9. Butt of KIA yellow
10. H2S production negative
11. Gas production negative
 Vibrio cholerae
 It has two species

 Eltor
 Vogues proskuer positive
 Resistance to polymixin B
1. Not lysis by phage
 Classical
 Vogues proskuer negative
 sensitive to polymixin B
 Biochemical reactions of psuedomonas:-
1. Lactose negative
2. Manitol negative
3. Glucose negative
4. Citrate positive
5. Motility positive
6. Indole negative
7. Urease sometimes
8. Slope of KIA red
9. Butt of KIA red
10. H2S production negative
11. Gas production negative
 This group contains:-
1. Yersinia
2. Haemophilus
3. Bordetella
4. Brucella
 All of them are oxidase negative except bordetella and
brucella
 Yerinia shows bipolar staining
 Haemophilus needs X and V factor to grow
 Brucella can grow on peptone water
 Bordetella and Haemophilus can not grow on peptone water
 Bordetella ferments glucose and lactose with acid
production
 Yersinia
 Blood agar:-
 Small shiny non haemolytic coloniea

 XLD,SS, and DCA

 Non lactose fermenting with no hydrogen sulfide production

 Macconkey
 Non lactose fermenting colonies
 Differentiation between Yersinia and other non
lactose fermenting enterobacteria

 No hydrogen sulfide or gas production to differentiate it from

Salmonella and Protues

 Urease positive to differentiate it from shigella

 Phenylalanine test is usefull to differentiate between yersinia

and protues

 The test is positive in Protues and negative in Yersinia

 Biochemical reactions :-
1. Lactose negative
2. Manitol positive
3. Glucose positive
4. Citrate negative
5. Motility positive
6. Indole sometimes
7. Urease positive
8. Slope of KIA red
9. Butt of KIA yellow
10. H2S production negative
11. Gas production negative
 Haemophilus influenzae
 The culture must contains:-
1. Porphyrin (haematin) (X factor)
2. Nicotinamide adenine dinuclotide or its phosphate
(NAD or NADP) as V factor

 X factor is used for the production of the respiratory

enzymes like catalase,cytochrome and peroxidase

 V factor is used as electron carrier in the oxidation

reduction system
 Not grow on lyzed blood agar because of the absence of factor
V
 Grow well on chocolate blood agar because the V factor will
appear when heating the RBCs
 Sometimes S.aurues produces factor V more than its need
 So we can inoculate it with H.influenzae to give factor V
 This forms the basis of the satellisim test which is the simplest
test used to differentiate between Haemophilus
 The organism is inoculated in N.A and B.A with S.arues
across it
 If the organism grow in the two medias it only needs factor V
(H.parainfluenzae)
 Brodetella
 Charcoal cephalexin blood agar is used as selective and
enrichment media.
 Produce small raised shiny colonies
 B.pertussis can not grow on blood agar
 Ferment glucose and lactose with acid production
 Oxidase positive
 Catalase positive
 Urease negative
 Can not grow on peptone water
 This groub include:-

1. Bacillus

2. Corynebacteruim

3. Clostridium
 Bacillus
 Catalase positive

 Spore forming

 Blood agar:-
 Large gray-white irregular colonies with wavy edges

 Manitol egg yolk phenol red polymyxin used as

selective media
 Biochemical reactions
 Gelatin stab culture:-

 The organism slowly liquefies the gelatin along the line of

culture

 No biochemical is used but it identified from the stain

appearance in the microscope
 Corynebacteruim
 The main type is Corynebacteruim Diphteriae
 It contains three biovars:-

 Gravis

 Mitis

 intermedius
 Tellurite blood agar
 Reduce tellurite and produce black-gray colonies

 Modified tinsdale meduim

 Gray-black raised colonies surronded by dark brown area due
to hydrogen sulfide production
 From the interaction between cystine and tellurite

 Dorset egg media

 Fast growth with formation of the granules
 Biochemical reactions
 CHO utilization
 The three of them utilize glucose and maltose but only gravis
utilize starch

 Elek gell preceptation

 To check the toxigenicity of the organism

 Schick test
 Skin test used to demonstrate the antitoxin produced in
response to infection
 Clostridium
 Neomycin blood agar is selective media
 C.perfringens on blood agar shows beta
haemolysis
 C.tetani on blood agar fine film of growth is
producwd
 Haemolysis may occur
 Biochemical reactions
 Lactose egg yolk milk agar used to detect:-

 Lecithinase C activity
 Appear as opacity in the medium due to lecithin breakdown

 Lipase hydrolysis
 Fatty layer covering the colonies and media
 Lactose fermentation
 Indicator is nuetral red

 Protinase activity
 Clearing around colonies due to breakdown of casein
 May need 72 hours of incubation

Prot LAC LIP Lecith.C species

- + - + Perfringens
D - + - Botulinum
- - - - Tetani
 Nagler reaction

 Inhibiting of the opacity caused by the lecithinase C

activity

 Adding antitoxic sera will inactivate the lecithinase C

activity
 Nisseria has two species:-

1. N.gonorrhoeae
2. N.meningitidis

 We differentiate between them by hiss serum sugar

Suc Mal Lact Glu Species

- - - + N.gonorrhoeae

- + - + N.meningitidis