General bacteriology revision notes microbiology

Microbiology
TONY SCARIA 2010 KMC

General microbiology

• Father of microbiology: louis Pasteur
• GREW BACTERIA IN LIQUID MEDIA
• Father of bacteriology : Robert Koch
• INTRODUCED SOLID MEDIA
• Father of chemotherapy : paul ehlrich
• Father of modern antiseptic surgery : john lister
• Used carbolic acid

Louis Pasteur
• Germ cell theory
• Developed anthrax rabies vaccine
• Developed sterilisation technique
• Sterilise,autoclave,hot air oven

Robert Koch
• Discovered tubercle bacilli (kochs bacillus) and vibrio cholera

Koch’s postulates
• 1. The microorganism must be present in every case of the but absent
from healthy host.
• 2. The suspected microorganism must be isolated and grown in a pure
culture from lesions of the disease.
• Except : Mycobacterium leprae and Treponema pallidum,rickettsiae ,many
viruses
• 3. The isolated organism, in pure culture, when inoculated in suitable
laboratory animals should produce a similar disease.
• 4. The same microorganism must be isolated again in pure culture
from the lesions produced in experimental animals.

Koch’s phenomenon
• hypersensitivity reaction against tuberculosis bacilli demonstrated in
guinea pigs.
• guinea pigs already infected with tubercle bacillus, on challenge with
tubercle bacillus or its protein, developed an exaggerated
inflammatory response

• GP+VE CELL WALL IS THICKER RETAINS PRIMARY STAIN SLOW
DECOLOURISATION
• GP+VE CELL WALL IS ACIDIC
• G N-VE HAS MORE LIPIDS  GET DISSOLVED BY DECOLORISER 
LEAD TO FORMATION OF PORES  RAPID DECOLOURISATION

Acid fastness
• Mycolic acid
• Integrity of cell wall

Acid fast staining
• Ziehl neelson staining
Smear stained by carbol fuchsin
Decolorisation by sulphuric acid
Counterstain with methylene blue
Retain fuchsin (red
colour)
Take colour of
methylene blue (blue
colour)
• Mycobacteria tuberculosis
(25%)
• Lepra (5%)
• Nocardia(1%)

Acid fast organism
• Bacteria
• Mycobacterium
• Nocardia
• Rhodococcus
• Legionella micdadei
• Oocyst of parsite
• Bacterial spores

Bipolar staining
• Safety pin appearance
• d/t accumulation of dye at
both ends

Bipolar staining
• Calymmobacter granulomatis
• Pseudomonas mallei
• Yersinia pestis
• Haemophilus ducreyi

GELATIN
• FIRST SLDIFYING AGENT
• PROTEIN IN NATURE
• REQUIRED @ CONCENTRATION 15 %
• FROM ANIMAL BONE
• DISADVANTAGE
• PROTEOLYSED BY BACTERIA LIQUEFACTION
• SOLID ONLY @ TEMP <24 *C

AGAR
• MOST COMMONLY USED
• POLYSACCHARIDE (AGAROSE (70%)+ AGAROPECTIN (30 %)+INORGANIC
PHOSPHATE
• 1.2- 2 % IS NEEDED  2 % CONCENTRATION IN SOLID MEDIA
• FROM SEA WEEDS & RED ALGAE  GELIDIUM
• INERT  NOT HYDROLYSED BY BACTERIA
• NO NUTRITION TO BACTERIA
• NO GROWTH PROMOTERS OR INHIBITORS
• SOLID @ TEMP <42 *C
• MELTS @ TEMP > 98 *C

SOFT AGAR
•0.2 – 0.5 %
•SEMISOLID
•FOR
BACTERIAL
MOTILITY
USUAL SOLID
MEDIA
•1.2- 2 %
5-6 % AGAR
•FORM AGAR
•FOR
INHIBITION
OF
SWARMING

SWARMING
GRAM POSITIVE
• CLOSTRIDIUM TETANIN
• BACILLUS CEREUS
GRAM NEGATIVE
• PROTEUS VAULGARIS
• PROTEUS MIRABILIS
• VIBRIO ALGINOLYTICUS
• VIBRIO PARAHEMOLYTICUS

OTHER SOLDIFYING AGENTS
• EGG
• SERUM

TYPES OF CULTURE MEDIA
SIMPLE MEDIA
• FOR
GROWING
NON
FASTIDIOUS
BACTERIA
• ONLY SOURCE
OF CARBON &
NITRIGEN
ENRICHED
MEDIUM
• MEDIUM
WITH EGG
BLOOD
SERUM
SELECTIVE
MEDIUM
• SELECTIVE
AGENT
ADDED TO
INHIBIT
OTHER
BACTERIA &
PROMOTE
WANTED
BACTERIA
Enrichment
media
• Liquid
selective
media
Differential
media
• Medium to
differentiate
b/w bacteria
• BA
• MacConkey
medium
Indicator media
• Indicates
bacterial
growth
• Tellurite
media

SIMPLE MEDIA
• PEPTONE + H20 PEPTONE BROTH
• PEPTONE BROTH + MEAT EXTRACT  NUTRIENT BROTH
• NUTRIENT BROTH + AGAROSE  NUTRIENT AGAR

ENRICHED MEDIA
• BLOOD
• 5% BA  FOR HEMOLYSIS DEMONSTRATION
• 10 % BA
• CHOCOLATE AGAR
• EGG CONTAINING
• LOWEN STEIN JENSON MEDIUM
• DORSET EGG MEDIUM
• SERUM
• LOEFFLERS SERUM SLOPE
• PPLO MEDIUM

SELECTIVE MEDIA
• Change pH
• Only for vibrios (pH 8.2 -8.4)
• Bile salt agar
• TCBS medium
• Adding abx
• Thayer martin medium  vancomycin +colistin +nystatin
• Modified Thayer martin  TM medium + trimethoprim
• New York city medium vancomycin + colistin + trimethoprim + amphotericin B
• PPLO with penicillin
• Adding chemical agents
• Bile salts 
• MacConkey  gram negative
• XLD selective for salmonella & shigella
• DCA selective for salmonella & shigella
• Tellurite  corynebacterial macLeod
• 10 % salt  staphylococci  salt agar saltmilk agar
Neisseria

• By adding dyes
• methylene blue agar  gram negative bacteria
• LJ media  malachite

Liquid selective broth
• Alakaline peptone for vibrios
• Tetrathionate broth only for salmonella
• Selenite F broth for salmonella & shigella

Differential media
• TCBS
• Thiosulfate
• Citrate
• Bile salt
• Sucrose

Differential media
• Mannitol salt agar
• Only mannitol
fermenting staph aureus

Hugh leifson OF mediumdifferential media

Petroleum jelly to make medium anaerobic

CLED media  for urinary pathogens

Indicator media
• Tellurite media for Corynebacterium
• Wilson blair media for salmonella

Transport media
• Donot allow growth of commensals
• Maintain pathogens
• Pikes medium  streptococcus pyogenes
• Thioglycolate broth for anaerobes
• Cary blair broth  universal stool transport media
• Stuarts media & amies media for Neisseria

• Salmonella, Shigella
from faecal specimens

ANEROBIC MEDIA
• ROBERTSONS COOKED MEAT MEDIA
• SMITH NOGUCHI MEDIUM
• HEATING & SUBSEQUENT PLATING MEDIA

ANAEROBIOSIS

Antibiotic susceptibility test

MUELLER HILTON AGAR
• STARCH + CASEIN HYDROLYSATE + AGAR
• ANTIBIOTIC SENSITIVITY TESTING

Antibiogram

Standard inoculum

0.5 Mcfarland  1.5 * 10 ^ 8 bacteria from 4-
5 colonies

• Inoculated in to millor Hilton agar @ 37 * c
• Interpreted @ 16- 18 hrs

Methods of antibiotic sensitivity
• Dilution best method  best method / reference mthod
• Exact Minimum inhibitory concentration can be determined
• Broth dilution
• Microbroth  microtitre plate
• Macrobroth  testtubes
• Agar dilution
• More time consuming 
• Kirby bauer disc diffusion method

Lowest concentration of Abx just inhibit bacterium
inmmedium  MIC (minimum inhibitory
concentration)

Microbroth

Kirby bauer
disc diffusion
method

Stokes method of disc diffusion

E STRIP METHOD
EPSILOMETER  E TEST (GRADED
CONCENTRATION OF ABX SHEET)

Bacterial growth curve

• Generation time  time required for 1 binary fission
• Viable count no of live bacteria
• total count  no live + Killed bacteria

Lag phase
• Stage for adaptation
• Variable for different bacteria
• Metabolically active
• Size of bacteria is maxmm @ end of lag phase
• No replication  count (viable & total) remains constant

Log phase / exponential
• Stage of active replication
• Viable count ↑
• Total count increase ↑
• Size decreases

Stationery phase
• Gradual nutrient depletion
• No of bacteria multiplying= no of bacteria dying
• Viable count constant
• Total count increases
• Sporulation toxin production occurs

Declining phase / death phase
• Total nutrient depletion
• Most bacteria are dying
• Viable count decreases
• Total count is constant

• Maximum effect of abx adhesion is maximum in log phase

Maximum cell size in
log phase
Cells are smaller stain
uniformly  used for
microbiological stains
• Spore formation
• Exotoxin production
• Antibiotic proudtion
Involutional forms are
seen

Types of culture
Batch culture
• With fixed amount of nutrients
• Bacterial growth curve present
• Closeed system
Continous culture
• Maintenance of log phase
• In chemostat or turbidistat
• Fresh nutrients added
• Toxic nutrients removed

Obligate
aerobes
• Grow Only in
aerobic
conditions
Facultative
anaerobe
• Prefer aerobic
condition but
can also grow
in anaerobic
condiions
Microaerophilic
anerobes
• Prefer
anaerobic
conditions but
can tolerate
low amount of
o2
Obligate
anaerobe
• Grow only in
anerobic
environment &
cannot
tolerate
aerobic
condition
Capnophilic
• Grows better
in 5-10 % CO2
• Brucella
abortus
Vibrio cholera Most pathogenic
bacteria
Campylobacter
Helicobacter Brucella abortus
Clostridium
Bacteriodes

Temperature requirement
Psychrophilic
• <20 *c
• Bacteria
responsible for
Spoiling of
referigertaed
foods
mesophilic
• 25 – 40 *c
• Most human
pathogens
Thermophilic
• High
temperature
• Indicator of
sterilization
• Cause spoilage
of canned foods

Spore formation
• NOT A METHOD OF REPRODUCTION
• In unfavourable condition
• SPORES ARE ACID FAST
• Destroyed by autoclaving

L form / lister forms

Resolution
• Eye  0.2mm
• Light microscope  0.2um
• Electron microscope  0.2nm

UREASE PRODUCING BACTERIA
• PUNCH
• PROTEUS MIRABILIS
• UREAPLASMA UREALYTICUM
• NOCARDIA
• CRYPTOCOCCUS
• HELOBACTER PYLORI

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