BACTERIOLOGY

Prokaryote vs. Eukaryote

Prokaryote
Nuclear body
Cell division
Cell wall
Cytoplasmic
membrane
Cell organelles
Site of energy
production
Site of protein
synthesis

Eukaryote

Bacterial Cytology
1. Cell envelope
a) Cell membrane

2.

b)

Cell wall

c)

Glycocalyx

Cell appendages
a) Flagella

b)

3.

Pili / fimbriae

Cytoplasmic Structures
a) Nucleoid
b)

Plasmids

c)

Ribosomes

d)

Inclusions / granules

e)

Endospores

Bacterial Growth
1. Reproductive method
2. Generation time
3. Phases of growth

Bacterial Morphology
1. Size
2.

Shape

Bacteriology Part I / Prepared by: Karen Krista M. Escobar

4.

Measurement of bacterial growth

a) Direct methods
i. Plate count
1

ii. Membrane filtration technique

iii. Most probable number
iv. Direct microscopic count
b)

a)

Indirect methods
i. Turbidimetry
ii. Metabolic activity
iii. Dry weight

Requirements for Bacterial Growth

1. Energy requirements
a) Phototroph
b) Chemotroph
2. Carbon requirements
a) Autotroph
b) Heterotroph
3. Oxygen requirements
a) Aerobe
b) Anaerobe
c) Facultative aerobe
d) Facultative anaerobe
e) Microaerophile

4.
5.

6.

7.

CO2 requirements
a) Capnophile
Temperature requirements
a) Psychrophile
b) Thermophile
c) Mesophile
pH requirements
a) Acidophile
b) Alkalinophile
c) Neutrophile
Ionic strength and osmotic pressure
a) Osmophile
b) Halophile

Microbial Control
Sterilization vs. Disinfection
Microbicidal vs. Microbiostatic
Methods of Microbial Control
1. Physical agents
Bacteriology Part I / Prepared by: Karen Krista M. Escobar

b)
c)
d)
e)
f)
g)

Heat
i. Moist heat
Boiling or flowing steam
Autoclaving
Tyndallization
Inspissation
Pasteurization
Batch
Flash
ii. Dry heat
Direct flaming
Incineration
Hot air sterilization
Cremation
Filtration
Cold
Desiccation
Osmotic pressure
Sonic vibration, trituration, agitation
Radiation
i. Ionizing
ii. Non-ionizing

2.

Chemical agents
a) Alcohols
b) Surface active agents
c) Phenols
d) Halogens
e) Heavy metals
f) H2O2
g) Aldehydes
h) Organic acids
i) Gases
j) Dyes

3.

Antibacterial agents
a) Inhibits cell wall synthesis
i. Penicillin
ii. Cephalosporins
iii. Monobactams
iv. Carbapenem
v. Cycloserine
vi. Vancomycin
vii. Bacitracin
viii. Novobiocin
b) Inhibits CHON synthesis
i. Aminoglycosides
ii. Tetracyclines
iii. Nitrofurans
iv. Chloramphenicol
v. Macrolides
c) Inhibits nucleic acid synthesis
2

d)

e)

f)

i. Rifamycin, rifampin
ii. Quinolones
Inhibits bacterial metabolism
i. Sulfonamides
ii. Sulfones
iii. PAS
iv. Trimethoprim
v. INH
vi. Ethambutol
Inhibits folic acid (folate)
i. Sulfonamides
ii. Trimethoprim-sulfamethoxazole (Co
trimoxazole)
Damages cell membrane
i. Polymixins
ii. Polyenes
iii. Azoles

*Limulus Lysate test

Laboratory Diagnosis
1.

Microscopic examination
a) Living state / unstained
i. Wet mount preparation
ii. Hanging drop preparation
b) Fixed state / stained

STAINING
1) Grams staining
VIAS-

Bacterial Genetic Transfer

1. Conjugation
2.

Transformation

3.

Transduction

All cocci are G (+) EXCEPT:

NVM-

Pathogenecity vs. Virulence

1.

Adherence factors

2.

Antiphagocytic factors

3.

Enzymes

4. Toxicity
Differences
Typical sources
Manner of
release
Chemical
composition
Toxicity
Lethal dose
Effects on hosts
tissues
Heat
denaturation
(60-80oC)
Immunology
Pharmacology
Sample Diseases

Exotoxin

Endotoxin

Bacteriology Part I / Prepared by: Karen Krista M. Escobar

All bacilli are G (-) EXCEPT:

MBCLKNRAPLECG (+) becomes G (-)
______________________________
______________________________
______________________________
______________________________
______________________________
G (-) becomes G (+)
______________________________
______________________________
3

Non-staining method:
a) LANA (L-alanine-4-nitroanilide)
b) Strings test
2)
Purpose

b)
c)

Acid-fast staining

Ziehl-Neelsen

Kinyouns

Pappenheim
s

Baumgartens

Auramine-rh
odamine

Primary stain

decolorizer

counterstain

result

Reporting:
3)

4)

Special staining
a) Capsule - HGAWTM
b)

Spore - DAWS

c)

Metachromatic granules - NALLBS

d)

Flagella - GLeF

e)

Nucleic acid - F

f)

Polar body - W

g)

Cell wall - D

h)

Rickkettsia - GM

i)

Spirochetes - L

j)

B. anthracis - M

Indirect staining (capsule)

a) India ink
b) Borris
c) Nigrossin

2. Culture methods
A culture medium usually consists of:
Types of Culture
a) Pure culture
i. Streak plate
ii. Pour plate
iii. Selective medium
Bacteriology Part I / Prepared by: Karen Krista M. Escobar

iv. Animal inoculation

Mixed culture
Stock culture

Classification of Culture Medium

a) Consistency
Broth/liquid
Agar/solid
Liquefiable
Non-liquefiable
Semi-solid
b) Chemical composition
Synthetic/defined
Nonsynthetic/undefined/complex
Tissue
c) Method of dispensing
Plated
Tubed
d) Function
Simple/primary/general purpose
media
Enrichment
Enriched
Differential
Selective
Inhibitors
Gram positive
Gram negative

Swarming

Inoculation techniques
a) Liquid/broth
b) Agar slant
c) Agar butt
d) Agar slant-butt
e) Plated medium
Methods of Streaking Plated Media
a) Simple streaking
b) Radial streaking
c) Overlap streaking
d) Multiple streaking
e) Interrupted streaking
f) Multiple interrupted streaking
Method of Cultivation
a) Aerobic
Examples of Culture Media
i. BHI broth/agar
4

Rapid LF

ii.

Late LF

Non LF

THIO
vi. TMA
vii. MTM
viii. MLA
ix. NYC agar
x.

GC agar

iii. BAP

xi. Phenyl ethyl alcohol agar

iv.

xii. Columbia CNA

CAP

xiii. BCA
xiv. Tinsdale Medium

v.

MAC

xv. MHA

Bacteriology Part I / Prepared by: Karen Krista M. Escobar

xx. SSA

xvi. MSA

xxi. BSA

xxii. BGA
xvii. EMB

xviii.XLD

xxiii.Tetrathionate broth

xix. HEA

Bacteriology Part I / Prepared by: Karen Krista M. Escobar

xxiv. TCBS
xxix. SSA

xxx. Selenite F

xxv. LJ

xxvi. Middlebrook 7H10/7H11

xxvii.

Ogawa

xxviii.

Trytic soy broth/agar

Bacteriology Part I / Prepared by: Karen Krista M. Escobar

xxxi. APW

xxxii.

CLED

xxxiii.

Cetrimide agar

xxxiv.

CHROM agar

xxxvi.

BCYE

xxxvii.

CIN agar

xxxviii.

Cystine tellurite agar

xxxix.

Regan lowe

xxxv.Bordet-Gengou agar
Bacteriology Part I / Prepared by: Karen Krista M. Escobar

xl. BEA

b)

Novobiocin resistant test

d)

Bacitracin susceptibility test

e)

Bacitracin-SXT test

f)

PYR test

g)

Vancomycin susceptibility test

Anaerobic
i. Anaerobic jars

ii.
iii.
iv.
v.
3.

c)

Anaerobic glove box or chamber

Roll tube method
Chopped meat medium
Thioglycollate medium

Biochemical tests

GRAM POSITIVE COCCI AND ANAEROBES

a) Catalase test

b)

Coagulase test

Bacteriology Part I / Prepared by: Karen Krista M. Escobar

h)

Bile exculin hydrolysis test

i)

Salt tolerance test

l)

CAMP test

m) Hippurate hydrolysis test

j)

k)

Optochin susceptibility test

Bile solubility test

Bacteriology Part I / Prepared by: Karen Krista M. Escobar

n)

Kanamycin Vancomycin Colistin test

KVC Pattern
i. RRR
ii. SSR
iii. SRS
iv. RSR
v. RRS

o)

DNA hydrolysis test

10

p)

Oxidase test

q)

Reverse CAMP test

r)

c)

ONPG test

d)

Carbohydrate fermentation test

a) TSI

b)

KIA

c)

RDA

GRAM NEGATIVE BACILLI

a) Nitrate reduction test

Nitrite reduction test (add zinc powder to

the nitrate negative)
b)

Gelatin hydrolysis

Bacteriology Part I / Prepared by: Karen Krista M. Escobar

11

e)

f)

b)

Methyl red test

c)

Vogues-proskauer test

d)

Citrate utilization test

LIA

IMVIC
a) Indole test
i. SIM

ii.

g)

Acetate utilization test

h)

Acetamide utilization test

MIO

iii. Rapid spot tests

Bacteriology Part I / Prepared by: Karen Krista M. Escobar

12

i)

Urease test

m) Oxidative fermentation test

j)

k)

Urea agar

n)

Butyrate disk

o)

MUG test

Decarboxylase / dihydrolase test

ID for both G (+) and G (-)

a) BBL crystal

b)

l)

API 20-E

Phenylalanine deaminase test

Bacteriology Part I / Prepared by: Karen Krista M. Escobar

13

Antimicrobial Susceptibility Test

Interpretation
SIR-

4.

Sputum culture

5.

Urine culture

6.

CSF

7.

Genital culture

8.

Stool culture

Tests methods
1. Agar-disk diffussion
2.

Broth dilution

3.

Agar dilution

4.

Colorimetric microdilution test

5.

Etest

Specimen Storage
1. 37oC
2. 4oC
3. -20oC
4. -70oC

6.

ESBL

Preservation of Organisms
1. Long term preservation
a) Best methods

b)

Alternative methods
i. Glycerol at -20oC

ii.
7.

AmpC

8.

Automated AST

iii. Stab culture at RT

Specimen Collection and Processing

1. Blood culture

iv.

2.
2.

Throat culture

3.

Nasopharyngeal culture

Bacteriology Part I / Prepared by: Karen Krista M. Escobar

Mineral oil at RT

Stab culture in CTA

Short term preservation

Life is a walk of faith to God, full of pressures yet

fulfilling, full of tests yet rewarding and
full of trials yet strengthening...
14