Bacterial Cultures in Broth Media

 Divided agar plate – semi-quantitative

Medium - If there is growth, last
quadrant magkuha.
a. Solid – indicates characteristics - 4 quadrants
b. Semi-solid – indicates motility
c. Liquid

Blood Agar

Shows three types of hemolysis:

 α hemolysis – indicates partial

hemolysis (RBCs)
 β hemolysis – indicates hemolysis
(RBCs)
 γ hemolysis – indicates no hemolysis
- bacteria incapable/ not
capable of hemolyze RBCs

 MacConkey Agar – pink colonies in lactose

fermenters

 Chocolate agar – blood is hemolyze

Antimicrobial Susceptibility Testing (AST) 2. Compare turbidity
3. Inoculate (stray) into MHA agar
4. Apply antibiotic disk and wait 3-5
minutes for inversion (baliktad)
5. Invert plates (MHA) and;
6. Incubate 37 degrees Celsius in the
incubator for 16-18 hours and next
morning go back and measure the zone
of inhibition
7. Interpret the susceptibility.
- based on the chart

 Direct measures of antimicrobial activity are POSSIBLE SOURCES OF ERROR

accomplished using: a) Used of mixed culture
1. Broth dilution b) Inoculum too light
2. Agar dilution c) Too heavy
3. Disk difusion d) Too much moisture on agar
 TUBE OR MICROPLATE DILUTION e) Very dry agar
(BROTH)
f) Improper storage of disk
Serial dilution of antibiotic is prepared, to each
tube, a uniform amount of inoculum is added g) Reading and clerical error

- Minimum Inhibitory Concentration (MIC) - the h) Deterioration of turbidity

lowest concentration of antibiotic that inhibits
bacteria
 Zone of inhibition (big) – sensitive
- Minimum Bactericidal Concentration (MBC) -
 Zone of inhibition (small) – resistant
lowest concentration of antibiotic that kills
bacteria

 AGAR DILUTION

-Varying concentration of antibiotic is

incorporated to appropriate plating media

 DISK DIFFUSION (KIRBY BAUER)

(MHA) MUELLER-HINTON AGAR (MHA): depth

4mm (3-5mm); pH 7.2-7.4

- STEPS (7)

SEVEN STEPS (MHA)

1. Pick 4-5 colonies and incubate in 37

degrees Celsius for 2-4 hours.
a. 0.5 McFarland -standard
turbidity (in 0.5)
i. Composition - 9.955 mL
of 1% H2SO4

- 0.5 mL of 1.175 BaCl2

ii. Inoculum (Bacteria)

- 1.5 x 108 CFU/mL

 FAMILY MICROCOCCACEAE

GENERA:

STAPHYLOCOCCUS

MICROCOCCUS

PLANOCOCCUS
 Mesophilic bacteria – clinically STOMATOCOCCUS
significant bacteria
LABORATORY
- can grow in 35- 37 degrees Celsius
1. Gram Stain
 E-Test (AST)
a) Each strip has a gradient of Staphylococcus = gram (+) COCCI in CLUSTERS
concentrations of a different (grape-like COCCI)
antimicrobial drug. Micrococcus = gram (+) COCCI in TETRADS
b) The MIC is determined by reading the (packets of 4/8/16)
number on the strip at the point at
which growth intersects the strip. 2. Growth on BAP

Staphylococcus =
 Automated (AST)
- machine for reading bacterial colonies  In blood agar plate
- creamy white, pinhead colonies
- has beta (β) hemolytic pattern

 α hemolysis – indicates partial

hemolysis (RBCs); greenish
 β hemolysis – indicates hemolysis
(RBCs); clearance
 γ hemolysis – indicates no hemolysis
 α-prime – if medium is from
refrigerator.
- small zone of α pattern (inner)
surrounded by a zone of β
Component of bacterial cell (left); Antibiotics
(right)
3. Growth on Loeffler's Serum Slant (LSS)  Modified oxidase test (Microdase)
LSS - long storage of bacteria for prolonged  Blueish discoloration – positive
use result
 S. aureus = golden-yellow colonies - one rapid test to differentiate
under LSS Staphylococcus from
 S. citreus = lemon-yellow colonies Micrococcus
 S. albus (epidermidis) = porcelain-white  Reagent – Tetramethyl
colonies paraphenylene diamine
dihydrochloride with DMSO
4. Growth on MSA (sel/diff media for Staph sp.)
(Dimethyl sulfoxide)
 Inhibitor = 7.5% -10% NaCl  CHO Oxidation Fermentation test (OF)
 CHO = Mannitol
 pH indicator = phenol red
ORGANISM OPEN CLOSED
TUBE TUBE
 S. aureus = can ferment; yellow color Staphylococcus Yellow Yellow
- can change Ph from red to yellow FERMENTER color color
 S. epidermidis = non-mannitol Micrococcus Yellow Green
fermenter OXIDIZER color color
- can change color to pink
 S. saprophyticus= variable (can change  CLOSED TUBE = sealed with
anytime; fermenter/ non-fermenter) VASPAR (VASELINE & PARAFFIN)
- color depends or MINERAL OIL PROPERTY
 CHO = glucose
5. Catalase test – differentiate streptococcus
 pH indicator =
and staphylococcus
 Bromthymol blue (BTB)
 Reagent – 3% hydrogen peroxide – if utilize become
 Effervescence/ bubbles – positive result acidic (yellow), alkaline
NOTE: don't use colony/inoculum from (green/blue)
BAP due to pseudoperoxidase activity of
hemoglobin o COMMON PH INDICATOR FOR
 STAPHYLOCOCCUS (+) OF TEST
 STREPTOCOCCUS (-)  Bromcresol purple – purple
to yellow
6. Tests to differentiate staphylococcus from
 Andrade’s acid fuchsin –
micrococcus
pale yellow to pink
TESTS STAPHYLOCOCCUS MICROCOCCUS  Phenol red – red to yellow
AEROBIC Growth Growth  Bromthymol blue – green to
GROWTH yellow
ANAEROBIC Growth No growth  Medium = CHO Oxidation
GROWTH Fermentation test (OF) medium
LYSOSTAPHIN Susceptible Resistant or tube
(antibiotic)
BACITRACIN Resistant Sensitive 7. Coagulase test
(antibiotic) - the most important pathogenic
MODIFIES Negative Positive determinant of S.AUREUS
OXIDASE TEST
GLUCOSE Fermenter Oxidizer A. Slide method
UTILIZATION - utilize glucose - with - detects the cell bound coagulase
with or without oxygen (clumping factor)
oxygen  Reagent: Rabbit plasma
(expensive)
- Alternative: Human plasma
(EDTA)
  Inoculum + rabbit plasma =  Results of the Voges-
Clumping Proskauer test resemble
B. Tube method those of the Methyl Red
- detects free coagulase test.
 Reagent – 0.5 mL rabbit plasma
 0.5 mL rabbit plasma +
8. DNAse test (DNA hydrolysis test)
inoculum = Gel-like fibrin clot
 Mixture – incubate in 37  CoNS=NOVOBIOCIN SUSCEPTIBILITY
degrees Celsius for 4 hours and TESTING
check every 30 minutes.  Medium – DNA medium with
- if after 4 hours no clot, add methyl green
another 20 hours (incubation)  If capable in hydrolysis, there is
- if after 20 hours no coagulant, clearance (clearing)
test is negative  DNAse (+):
o Staphylococcus aureus
DIFFERENTIATION AMONG COAG. + o Moraxella
STAPHYLOCOCCUS o Serratia

NOTE: All bacillus are gram-negative except

ORGANISM TUBE VOGES- PY Proteus, Providencia, Morganella
COAGULAS PROSKAUE R
E R  CoNS – coagulase negative
S. aures + + - Staphylococcus
subsp. - Novobiocin susceptibility testing
aureus
S. Variable - +  Novobiocin – uses 5 microgram of
intermediu novobiocin
s o Staph. epidermidis – causative
S. hyicus Variable - - agent of bacterial endocarditis
S. scleiferi + + + - in artificial valves
subsp.
- Susceptible: to novobiocin
coagulans
- has zone of inhibition/
clearance (14mm),
 PYR TEST (L-Pyrrolidonyl-ẞ- - if dli kaabot og 14mm
naphthylamide test) is a biochemical (resistant)
test used to detect the ability of
bacteria to produce pyrrolidonyl o Staph. saprophyticus – common
aminopeptidase enzyme. cause of UTI among many active
young females
 Voges-Proskauer test is used to - Resistant: to novobiocin
determine if an organism produces
PATHOGENIC DETERMINANTS OF S.AUREUS
acetylmethyl carbinol from glucose
fermentation.  protein A – inhibits phagocytosis
o Voges-Proskauer Test Results  coagulase – coats neutrophil to inhibit
 Ability of bacteria to phagocytosis
convert glucose to acetoin  staphylokinase (fibrinolysin) – dissolves
(diols and diones) creates a fibrin clots and may enable the infection
red/purple color near top of to spread
tube (a positive Voges-  lipase – hydrolyze lipids and plasma in
Proskauer result). skin (ex. Boils)
 Bacteria producing a  hyalurodinase – hydrolyse hyaluronic
negative Voges-Proskauer acid (in the tissue, skin)
result exhibit no color - also known as Duran-Renal
change. Factor
  Duran-Renal Factor – usually in - Clinically significant
joints (synovial fluid) - associated with neonatal
o S. aureus – predominant infection
pathogen in joints - can be seen in vagina,
pregnant women
 DNAse – degrades DNA
 Exofoliatins- hydrolyze tissue through o Group C - Clinically significant
cleavage of Statum granulosum - only SXT (Sulfamethoxazole)
If tissue cleaves: susceptible
Staphylococcal Scalded Skin - S. equisimilis
Syndrome (SSSS) – also known - S. zooepidemicus
as Ritte Lyel Disease - S. equi
- S. dysagalactiae
 LEUKOCIDINS – lysis neutrophils and
macrophage - Susceptible to:
 hemolysin – lysis RBCs Sulfamethoxazole &
 enterotoxins – Trimethoprim
 Enterotoxin A & B – for food poison - Causes:
 Enterotoxin F – for toxic shock  Pharyngitis
syndrome in skin (usually in palms  Bacteremia
or soles) - Considered opportunistic
- Associated with pneumonia,
cellulitis & abscess
FAMILY STREPTOCOCCACEAE

• GRAM POSITIVE o Group D - Clinically significant

• NONMOTILE - Enterococci and non-
• NON-SPORE FORMING enterococci
• FACULTATIVE ANAEROBES
Enterococci Non-
• CATALASE NEGATIVE
enterococci
Can grow Cannot grow
2 TYPES OF CLASSIFICATION (6.5% NaCl (6.5% NaCl
 LANCEFIELD CLASSIFICATION saline) saline)
-based on antigenic characteristic of a
group-specific cell wall polysacc. Ex:
 Group A – W -E. faecalis
o Group A – Streptococcus -E. faecium
pyogenes -E. durans
- Clinically significant -E avium
- Causative agent for:
 Pharyngitis -Causative agent for: UTI &
 Tonsilitis wound infection
 Scalet fever
 Impetigo NOT LANCEFIELD
 Cellulitis  Streptococcus pneumonia (Lobar
 Erysipelas prime)
 Necrotizing fasciitis – - catalase negative
flesh-eating bacteria - lancet shaped bullet
 ARF/ Acute rheumatic  Viridans Streptococci (Subacute
fever – ASCHOFF bodies bacterial endocarditis)
 Acute Glomerular - catalase positive
Nephritis 1. S. mutans
2. S. mitis
o Group B – Streptococcus 3. S. uberis
agalactiae 4. S. sanguis (S. sanguinis)
 5. S. salivarius  Positive for this test:
6. S. constellatus o Listeria monocytogenes
7. S. intemedius o Gardnerella vaginalis
o Campylobacter jejuni

 BROWN'S CLASSIFICATION 8. BACITRACIN AND SXT SUSCEPTIBILITY TESTS

-according to the pattern of hemolysis ORGANISM BACITRACIN SXT NOTES
- discovered by Brown & Smith (Taxo__)
0.04 ug
- Group A, B, C, D A (S. Susceptible resistant PYR
pyogenes) &
sensitive
ALPHA bile esculin Streptococcus
- 10mm
hydrolysis (+) pneumonia, viridans,
of zone of
some group D
inhibition
BETA Group A, B, C B (S. Resistant resistant  CAMP (+)
Some group D agalactiae)  Hippurate
GAMMA Most group D
(+)
C Resistant Susceptible

LABORATORY TEST D resistant resistant  Bile

ENTEROCO esculin (+)
1. GRAM STAIN CCI
 PYR (+)
 Staining – chain-like  Can grow
 Under gram-stain – lancet or bullet to 6.5%
shape NaCl
D resistant resistant  Bile
NON- esculin (+)
2. BAP (Blood agar plate) ENTEROCO
 Streptococcus – white pinpoint with CCI  PYR (-)
characterize hemolytic pattern  Cannot
grow to
6.5% NaCl
3. CATALASE – negative

4. BILE ESCULIN HYDROLYSIS -to determine the S. Viridans

ability to grow 40% bile & esculin hydrolysis pneumoniae
Mouse + -
5. PYR – also known as “P- virulence - (dead si lab.
dimethylaminocinnamaldehyde” Mouse)
- red color Inulin + -
- Group A & D (Enterococci) fermentation - (ma-ferment
- color developer si inulin)
Bile solubility + -
- (ma-lyse ang
6. CAMP REACTION
colonies)
- discovered by Christine-Alkins-Munch-
Optochin Sensitive Resistant
Petersen (TAXO__) -10 mL of
- used to differentiate beta-hemolytic Chemical zone of
-enhance hemolytic reaction name: Ethyl inhibition
- positive arrowhead zone of hemolysis hydrocuoreine
- Group B (Beta) HCI
Neufeld + -
7. Hippurate hydrolysis Quellung - (capsule
- detects hydrolysis of sodium Hippurate swelling)
into benzoic acid & glycine
 Positive reaction – purple  Erysipeloid – caused by Erysipelothrix
 Glycine + Ninhydrin = purple rhuseopathiae
 - handling of raw fish - binds fibrinogen blocking the
complement pathway activation
 Complement pathway –
frontline defense in the immune
system

 Scarlet fever – affects children in ages 5-

15 years old Types of Complement pathway:
- also known as “Scarlatina” 1. Classical pathway
 Susceptibility test – Dick’s test 2. Alternative pathway
- Arm + Erythrogenic toxin = 3. Mannan-binding pathway
redness (red) or erythema
 Diagnostic test – Shcultz- NOTE: All strains of Strep. produce C5a
Charlton peptidase
- Arm + Antitoxin = (+)  C5a peptidase – inactivate
neutralization / “bleaching chemotactic capability of
phenomena” neutrophil, monocyte, or WBC
that is capable of phagocytosis
 Rheumatic fever with ASHCHOFF bodies
– developed when infected by
Streptococcus. Antigenetic determinant of Group A Hemolysis

STREPTOLYSIN O STREPTOLYSIN S
Procedure for CAMP test Oxygen labile Oxygen stable
• Standard Method - destroys if oxygen is
present
 Firstly, make a single straight line streak of
Antigenic Nonantigenic
beta-hemolysin producing Staphylococcus
Responsible to Surface hemolysis
aureus down the center of a blood agar
subsurface hemolysis
plate,
 Then, inoculate a streak of the test
organism (beta-hemolytic streptococci to be NOTE:
identified) perpendicular to the
 S. pyogenes – large zone of hemolysis
staphylococcal streak. Take care not to
 S. agalactiae - small zone of hemolysis
intersect the staphylococcal streak.

Other method: disk method, spot rapid method Gram-negative cocci

 Reverse CAMP test

- This test is used for the
differentiation of Clostridium
perfringens from other Clostridium
species.
- In this method, the CAMP factor
produced by S. agalactiae is used to
detect the Clostridium perfringens
from other Clostridium species,
that's why this method is termed as
areverse CAMP test.
o S. pyogenes – is a powerful modulate
(balance or control) of immune system.
- has M protein (antigenic determinant)
Under:
 M protein – can bind to factor H
(important to complement  Nesseria and Moraxella catarrhalis
pathway)
Characteristics:
  Gram-negative diplococci
(kidney-shaped)
- except N. elongate (elongated
shape)
 Obligate aerobes – need oxygen

 Capnophilic – need 5-10% carbon GLUCO MALTO SUCRO LACTO

dioxide SE SE SE SE
 non-motile M. - - - -
 Catalase positive (+) – CPOP catarrhalis
- all except N. elongata N. + - - -
gonorrhoe
 Oxidase positive (+) – CPOP
ae
Gram-negative cocci – establish infection N. + + - -
through attachment of fimbriae or pilus meningitid
is
- All are normal flora except N. gonorrhoeae & N. subflava + + +/- -
N. meningitidis N. + + - +
lactamica
N. gonorrhoeae N. meningitidis
- causative agent of
gonorrhea  Dracon swab & Rayon-Tripped –
- found in genital preferred swab
tract or genitourinary  JEMBEC, Bio-bag, Geno-pak, Transglow
tract  Acute – Commercial media for Neisseria
- transmitted through purulent
 0.025% SPS – anticoagulant
sexual intercourse meningitis
preservation that is for Neisseria
- life-
 Ophthalmia threatening  35-37 degrees Celsius – fastidious
neonatorum - rapidly bacteria (ex. Neisseria)
(of newborn) progressive bacterial  Ureaplasma urealyticum &
- cause of infection of the Mycoplasma hominis – can grow in
conjunctivitis meninges and New York City agar
in newborns subarachnoid space.  N. meningitidis & N. catarrhalis – can
 Optimysin or  Waterhouse- grow in 5% blood agar
silver nitrate Friderichsen
– remedy of Syndrome
conjunctivitis -
in neonates hemorrhage
or newborns in adrenal
gland

 Other Neisseria spp. – causes

endocarditis, meningitidis, bacteremia