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HOST-MICROBE RELATIONSHIPS &
DISEASE PROCESSES
by
Ayesha Fatima
Learning Outcomes
• To understand the establishment of Koch’s Postulates in
relation to infectious diseases.
• To understand the relationship between normal microbiota
and host.
• To study disease patterns, spread of infections and
transmission.
• To identify nosocomial infections & their transmission.
• To learn to isolate cultures from clinical samples & perform
antimicrobial sensitivity tests.
• To interpret the mechanisms of pathogenicity.
2
Introduction
• Pathology – the scientific study of disease.
• Pathogen : a microorganism that is able to produce
disease.
• Pathogenicity is the ability of a microorganism to
cause disease in another organism, namely the
host for the pathogen. May be a manifestation of a
host-parasite interaction
• Etiology – the cause/agent of a disease.
3
• Infection – the invasion & growth of pathogens in the
body.
• Disease – an abnormal state in which parts/ all of
body is not properly adjusted or is incapable of
performing normal functions.
• Normal microbiota – microorganisms that establish
permanent colonies inside or on the body without
producing disease make up the normal microbiota.
• Transcient microbiota – microbes that are present for
various periods and then disappear.
• Signs – changes that can be observed by the
physician; e.g. lesions, fever, swelling.
• Symptoms – changes in the body function
observed by the patient; e.g. body pain &
discomfort.
• Diagnosis – the process of determining the
nature of a disorder by considering the
patient’s signs & symptoms; e.g. using lab test
results, X-ray.
5
Anatomical Location Predominant bacteria
Skin staphylococci and corynebacteria
Conjunctiva sparse, Gram-positive cocci and Gram-negative
rods
Oral cavity
teeth streptococci, lactobacilli
mucous membranes streptococci and lactic acid bacteria
Upper respiratory tract
nares (nasal
membranes)
staphylococci and corynebacteria
pharynx (throat) streptococci, neisseria, Gram-negative rods and
cocci
Lower respiratory tract none
Our guests
Our guests
Gastrointestinal tract
stomach Helicobacter pylori (up to 50%)
small intestine lactics, enterics, enterococci
colon bacteroides, lactics, enterics, enterococci, clostridia,
methanogens
Urogenital tract
anterior
urethra
sparse, staphylococci, corynebacteria, enterics
vagina lactic acid bacteria during child-bearing years;
otherwise mixed
(a)Bacteria on skin surface, (b)Plaque on
enamel, (c)Bacteria of large intestines
8
Symbiotic relationship with microbes
Mutualism
Both organisms
benefit
Commensalism
One organism
benefits, other
unaffected
Parasitism
Host is
harmed
while the
guest
benefits
Lactobacilli on
vaginal epithelial
cell
S epidemidis on
skin cell
Pseudomonas
aeruginosa
causes several
infections
Symbiotic relationships
• Mutualism –E.g. The bacteria are provided with a constant temperature
and supply of nutrients (glycogen) in exchange for the production of
lactic acid, which protects the vagina from colonization and disease
caused by yeast and other potentially harmful microbes.
• Commensalism – our relationship with Staphylococcus epidermidis, a
consistent inhabitant of the skin of humans. Probably, the bacterium
produces lactic acid that protects the skin from colonization by
harmful microbes that are less acid tolerant. But it has been suggested
that other metabolites that are produced by the bacteria are an
important cause of body odors (good or bad, depending on your
personal point of view) and possibly associated with certain skin
cancers.
• Parasitism – The mode of existence of a parasite implies that the
parasite is capable of causing damage to the host. Some parasitic
bacteria live as normal flora of humans while waiting for an
opportunity to cause disease Eg. Members of the normal flora such as
Staphylococcus aureus or E. coli can cause an opportunistic infection.
10
Other Host-Microbe Relationships
• Opportunistic pathogens – pathogens that don’t
cause disease under normal conditions but cause
disease under special conditions. Eg. E coli,
Streptococcus pneumonia, Haemophilus influenzae
• Microbial Antagonism – provides competition for
other microbes. E.g E.coli in the intestines prevents
other microorganisms from causing infection
11
Koch’s Postulates
• The same pathogen must be present in every case
of the disease.
• The pathogen must be isolated from the diseased
host and grown in pure culture.
• The pathogen form pure culture must cause the
disease when it’s inoculated into a healthy,
susceptible laboratory animal.
• The pathogen must be isolated from the inoculated
animal and must be shown to be the original
organism.
12
Koch’s Experiment
13
Exceptions to Koch’s Postulates
• Modified to establish etiologies of diseases caused
by viruses and some bacteria, which cannot be
grown on artificial media.
o Some diseases, such as tetanus have unequivocal signs
& symptoms.
o Some diseases such as pneumonia may be caused by a
variety of microbes. E.g. Streptococcus pneumonia and
Haemophilus influenzae
o Some pathogens such as Streptococcus pyogenes cause
several different diseases.
o Streptococci cause tonsillitis (strep throat), pneumonia,
endocarditis. Some streptococcal diseases can lead to
rheumatic fever or nephritis which can damage the heart
and kidney E.g.
o Certain pathogens, such as HIV cause disease in
humans only.
14
Classification of Infectious Diseases
• Communicable diseases – are transmitted directly or
indirectly from one host to another.
• Non-communicable diseases – caused by
microorganisms that normally grow outside the
human body & are not transmitted from one host to
another.
• Contagious diseases – one that is easily spread from
one person to another.
15
Severity/ Duration of disease
• Acute disease – disease that develops rapidly but
lasts only for a short period of time.
• Subacute – an intermediate between acute &
chronic.
• Chronic - disease that develops more slowly, but
likely to be continual or recurrent for long periods.
• Latent – a disease in which the causative agent
remains inactive for a period & then becomes active.
16
Types of Infections
• Local Infection – microbial infections are limited to
one region of body. E.g. boils.
• Systemic Infection – microbial products can spread
to other parts via lymphatic system or bloodstream.
• Focal infection - Systemic infection that began as a
local infection.
• Primary Infection – acute infection that causes the
initial illness.
• Secondary Infection – caused by opportunistic
pathogens after primary infection.
17
Frequency of Occurrence
• Incidence – the no of people in a population who
develop a disease during a particular time period;
an indicator of the spread of a disease.
• Prevalence – the no of people in a population who
develop a disease at a specified time, regardless
of when it started (how long or serious a disease in
that population).
• Epidemic – a disease acquired by many hosts in a
given area in a short time.
• Endemic – a disease that is constantly present in a
certain population.
• Pandemic – an epidemic that occurs worldwide.
• Sporadic – a disease that occurs occasionally in a
population.
18
Patterns of Diseases
• Predisposing factors – age,
gender, etc.
• Stages of disease –
(i) Incubation period
(ii) Prodromal period
(iii) Period of illness
(iv) Period of Convalescence
(v) Period of Decline
19
Development of Diseases
• Incubation period – time interval between the
initial infection & the first appearance of any
signs or symptoms.
• Prodromal period – short period, follows
incubation; early, mild symptoms of diseases;
e.g. general aches.
• Period of illness – most acute; exhibits overt
signs & symptoms; e.g. fever, muscle pain, sore
throat, etc.
20
Development of Disease
• Period of Decline – signs & symptoms subside;
fever drops, aches diminishes. Patient is
vulnerable to secondary infections.
• Period of Convalescence – patient regains
strength & body returns to normal state.
Recovery period. Can serve as reservoir of
disease & easily spread infection to others.
21
Reservoirs of Infections
• Human reservoirs – people who have a disease or
are carriers of pathogenic microbes.
• Animal reservoirs – wild & domestic animals that
can transmit disease to humans.
• Non-living reservoirs – soil, water, equipments.
22
Transmission of Disease
(A) Contact transmission
• Direct contact – direct transmission between the
source & a susceptible host. E.g. touching, kissing,
sexual intercourse - anthrax, AIDS.
• Indirect contact - agent of disease is transmitted
from its reservoir to a susceptible host by means of
nonliving object (fomite). E.g. tissues, beddings,
thermometer - hepatitis B, tetanus.
• Droplet transmission – spread of microbes in droplet
nuclei in short distance i.e. less than 1m.
E.g. Coughing, sneezing - influenza, pneumonia.
23
Contact transmission
24
Disease Transmission
(B) Vehicle Transmission
– Transmission of disease by a medium, i.e.
water, food, air.
– Waterborne – pathogens are spread by
contaminated water, poorly treated sewage.
E.g. cholera, shigellosis.
– Foodborne – pathogens are spread by food
improperly cooked, poorly refrigerated, are
prepared under unsanitary conditions. E.g.
food poisoning, tapeworm infections.
– Airborne – pathogens are spread by dust
particles that travel in air more than 1m in
distance. 25
(C) Vectors
– Animals that carry pathogens from one host
to another, E.g. arthropod & other insects.
– Mechanical transmission – passive transport
of the pathogens on the body parts of the
vectors that makes contact with host’s food.
– Biological transmission – active process in
which vectors bite an infected person and
pathogens multiply in vector and spreads to
another host after biting the host.
26
Disease Transmission
Nosocomial Infections
• Any infection that is acquired during the course
of stay in a hospital, nursing home or other
healthcare facilities.
• About 5-15% of all hospitalised patients.
• Normal microbiota can be a causative agent.
• Opportunistic pathogens, drug-resistant gram
negative & gram positive bacteria.
• Patients with burns, surgical wounds &
suppressed immune system.
• Transmitted by direct contact & fomites.
• Control by aseptic techniques, proper cleaning,
storage, handling of equipments & supplies by
hospital staff.
27
Factors of contribution to nosocomial
infections
28
Modes of disease transmission in a
hospital
29
Isolation of Clinical Samples
• Important to diagnose a disease.
• Samples to be taken aseptically.
• Container with sample should be labeled – patient’s
name, room number, date, time, medications taken.
• Must be sent to lab immediately.
• Cultured on selective or differential media to isolate &
identify any abnormal microbes.
30
Isolation of Clinical Samples
• Precautions are to be taken by all healthcare
workers, including students. (Refer to Appendix B
of text book).
• Instructions for taking clinical samples are specific
from one culture to another of different types.
• Wound/ Abscess, Ear, Eye, Blood, Urine, Faecal,
Sputum.
31
Antibiotic Sensitivity Testing
AST
Disc
Diffusion
Method
Kirby-
Bauer disc
diffusion
Stokes
disc
diffusion
Dilution
Method
Broth
dilution
Agar
dilution
(A) Medium used
• Mueller-Hinton agar or broth for aerobic
& facultative anaerobic isolates.
• Petri dish of size 100 mm in diameter &
depth of 4 mm (25 mL).
• pH of medium btw 7.2 and 7.4.
33
(B) Inoculum
• An overnight incubation is ideal.
• Then about 5-10 colonies are removed
and cultured in a broth culture for 4-6 hrs
(log phase).
• Density of organism should be appx. 108
cfu/ml.
• Inoculum of a standard strain should be
prepared as control.
34
(C) Antibiotic discs
• 6mm in diameter commercial discs with
accurate antibiotic concentration.
• Discs prepared in the lab should contain
proper diluted antibiotic solution.
• Discs and discs dispensers should be
stored in a sealed container at -20°C
(long term) or <8°C (immediate use).
• About 7 discs on a 100 mm plate.
35
Kirby-Bauer Disk
Diffusion Method
• An agar plate is swabbed 3 times with an
inoculum suspension.
• Lid is replaced and allowed to dry for 15
mins.
• Antibiotic discs are applied using sterile
forceps/ multidisc dispenser.
• Distance between one disc & another should
not be closer than 24 mm centre to centre.
• Plates are incubated at 35-37°C, 16-18 hrs.
36
Stokes Disc
Diffusion method
• Plate is divided into 3 parts: test organism
in the centre, control on the upper and
lower thirds OR control in the centre, test
organisms in the other 2 parts.
• An uninoculated gap of 2-3 mm to separate
the test & control area.
• A maximum of 6 antibiotic discs on a 100
mm plate.
• Incubated at 35-37°C, 16-18 hrs.
37
The principle of antibiotic diffusion in
agar
• As soon as antibiotic
impregnated disc comes
in contact with moist
agar surface, it absorbs
moisture from agar.
• Antibiotic diffuses into
the surrounding medium.
• As the distance from
disc increases, there’s a
logarithmic reduction in
antibiotic concentration.
• Reference strains should
be tested for each new
batch using S. aureus,
E. coli & P. aeruginosa.
38
Interpretation of inhibition zones
Eg. Chloramphenicol
Resistant Intermediate Susceptible
≤ 12 mm 13-17 mm ≥ 18 mm
39
Minimum Inhibitory Concentrations
(MIC)
• Is the least amount of antimicrobial agent
that inhibits visible growth of an organism
after overnight incubation.
40
Minimum Bactericidal Concentrations
(MBC)
Is the least amount of antimicrobial agent
that prevents bacterial growth after
subculture of the organism into an
antimicrobial-free medium.
MIC
• MIC can be determined in liquid or solid medium
(Mueller-Hinton).
• Antimicrobial agents of concentrations 0.25-125
µg/ml or other test concentrations are used.
• For broth dilution, final inoculum should be 105
cfu/ml & on agar about 1-2 µL is used.
• It should deliver about 104 cfu/spot.
• Incubation is at 35-37°C, 16-18 hrs (depends on
microbe).
• The lowest concentration at which there is no
visible growth is the MIC.
41
MBC
• Subculture is done from the tubes to a
NB free from antimicrobial agents.
• Incubated at 35-37°C, 16-18 hrs
(depends on microbes).
• Growth is examined.
• The tube that contains the lowest
concentration that fails to yield growth on
subculture is the MBC.
42

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PP 141 - L2a.pptx

  • 1. HOST-MICROBE RELATIONSHIPS & DISEASE PROCESSES by Ayesha Fatima
  • 2. Learning Outcomes • To understand the establishment of Koch’s Postulates in relation to infectious diseases. • To understand the relationship between normal microbiota and host. • To study disease patterns, spread of infections and transmission. • To identify nosocomial infections & their transmission. • To learn to isolate cultures from clinical samples & perform antimicrobial sensitivity tests. • To interpret the mechanisms of pathogenicity. 2
  • 3. Introduction • Pathology – the scientific study of disease. • Pathogen : a microorganism that is able to produce disease. • Pathogenicity is the ability of a microorganism to cause disease in another organism, namely the host for the pathogen. May be a manifestation of a host-parasite interaction • Etiology – the cause/agent of a disease. 3
  • 4. • Infection – the invasion & growth of pathogens in the body. • Disease – an abnormal state in which parts/ all of body is not properly adjusted or is incapable of performing normal functions. • Normal microbiota – microorganisms that establish permanent colonies inside or on the body without producing disease make up the normal microbiota. • Transcient microbiota – microbes that are present for various periods and then disappear.
  • 5. • Signs – changes that can be observed by the physician; e.g. lesions, fever, swelling. • Symptoms – changes in the body function observed by the patient; e.g. body pain & discomfort. • Diagnosis – the process of determining the nature of a disorder by considering the patient’s signs & symptoms; e.g. using lab test results, X-ray. 5
  • 6. Anatomical Location Predominant bacteria Skin staphylococci and corynebacteria Conjunctiva sparse, Gram-positive cocci and Gram-negative rods Oral cavity teeth streptococci, lactobacilli mucous membranes streptococci and lactic acid bacteria Upper respiratory tract nares (nasal membranes) staphylococci and corynebacteria pharynx (throat) streptococci, neisseria, Gram-negative rods and cocci Lower respiratory tract none Our guests
  • 7. Our guests Gastrointestinal tract stomach Helicobacter pylori (up to 50%) small intestine lactics, enterics, enterococci colon bacteroides, lactics, enterics, enterococci, clostridia, methanogens Urogenital tract anterior urethra sparse, staphylococci, corynebacteria, enterics vagina lactic acid bacteria during child-bearing years; otherwise mixed
  • 8. (a)Bacteria on skin surface, (b)Plaque on enamel, (c)Bacteria of large intestines 8
  • 9. Symbiotic relationship with microbes Mutualism Both organisms benefit Commensalism One organism benefits, other unaffected Parasitism Host is harmed while the guest benefits Lactobacilli on vaginal epithelial cell S epidemidis on skin cell Pseudomonas aeruginosa causes several infections
  • 10. Symbiotic relationships • Mutualism –E.g. The bacteria are provided with a constant temperature and supply of nutrients (glycogen) in exchange for the production of lactic acid, which protects the vagina from colonization and disease caused by yeast and other potentially harmful microbes. • Commensalism – our relationship with Staphylococcus epidermidis, a consistent inhabitant of the skin of humans. Probably, the bacterium produces lactic acid that protects the skin from colonization by harmful microbes that are less acid tolerant. But it has been suggested that other metabolites that are produced by the bacteria are an important cause of body odors (good or bad, depending on your personal point of view) and possibly associated with certain skin cancers. • Parasitism – The mode of existence of a parasite implies that the parasite is capable of causing damage to the host. Some parasitic bacteria live as normal flora of humans while waiting for an opportunity to cause disease Eg. Members of the normal flora such as Staphylococcus aureus or E. coli can cause an opportunistic infection. 10
  • 11. Other Host-Microbe Relationships • Opportunistic pathogens – pathogens that don’t cause disease under normal conditions but cause disease under special conditions. Eg. E coli, Streptococcus pneumonia, Haemophilus influenzae • Microbial Antagonism – provides competition for other microbes. E.g E.coli in the intestines prevents other microorganisms from causing infection 11
  • 12. Koch’s Postulates • The same pathogen must be present in every case of the disease. • The pathogen must be isolated from the diseased host and grown in pure culture. • The pathogen form pure culture must cause the disease when it’s inoculated into a healthy, susceptible laboratory animal. • The pathogen must be isolated from the inoculated animal and must be shown to be the original organism. 12
  • 14. Exceptions to Koch’s Postulates • Modified to establish etiologies of diseases caused by viruses and some bacteria, which cannot be grown on artificial media. o Some diseases, such as tetanus have unequivocal signs & symptoms. o Some diseases such as pneumonia may be caused by a variety of microbes. E.g. Streptococcus pneumonia and Haemophilus influenzae o Some pathogens such as Streptococcus pyogenes cause several different diseases. o Streptococci cause tonsillitis (strep throat), pneumonia, endocarditis. Some streptococcal diseases can lead to rheumatic fever or nephritis which can damage the heart and kidney E.g. o Certain pathogens, such as HIV cause disease in humans only. 14
  • 15. Classification of Infectious Diseases • Communicable diseases – are transmitted directly or indirectly from one host to another. • Non-communicable diseases – caused by microorganisms that normally grow outside the human body & are not transmitted from one host to another. • Contagious diseases – one that is easily spread from one person to another. 15
  • 16. Severity/ Duration of disease • Acute disease – disease that develops rapidly but lasts only for a short period of time. • Subacute – an intermediate between acute & chronic. • Chronic - disease that develops more slowly, but likely to be continual or recurrent for long periods. • Latent – a disease in which the causative agent remains inactive for a period & then becomes active. 16
  • 17. Types of Infections • Local Infection – microbial infections are limited to one region of body. E.g. boils. • Systemic Infection – microbial products can spread to other parts via lymphatic system or bloodstream. • Focal infection - Systemic infection that began as a local infection. • Primary Infection – acute infection that causes the initial illness. • Secondary Infection – caused by opportunistic pathogens after primary infection. 17
  • 18. Frequency of Occurrence • Incidence – the no of people in a population who develop a disease during a particular time period; an indicator of the spread of a disease. • Prevalence – the no of people in a population who develop a disease at a specified time, regardless of when it started (how long or serious a disease in that population). • Epidemic – a disease acquired by many hosts in a given area in a short time. • Endemic – a disease that is constantly present in a certain population. • Pandemic – an epidemic that occurs worldwide. • Sporadic – a disease that occurs occasionally in a population. 18
  • 19. Patterns of Diseases • Predisposing factors – age, gender, etc. • Stages of disease – (i) Incubation period (ii) Prodromal period (iii) Period of illness (iv) Period of Convalescence (v) Period of Decline 19
  • 20. Development of Diseases • Incubation period – time interval between the initial infection & the first appearance of any signs or symptoms. • Prodromal period – short period, follows incubation; early, mild symptoms of diseases; e.g. general aches. • Period of illness – most acute; exhibits overt signs & symptoms; e.g. fever, muscle pain, sore throat, etc. 20
  • 21. Development of Disease • Period of Decline – signs & symptoms subside; fever drops, aches diminishes. Patient is vulnerable to secondary infections. • Period of Convalescence – patient regains strength & body returns to normal state. Recovery period. Can serve as reservoir of disease & easily spread infection to others. 21
  • 22. Reservoirs of Infections • Human reservoirs – people who have a disease or are carriers of pathogenic microbes. • Animal reservoirs – wild & domestic animals that can transmit disease to humans. • Non-living reservoirs – soil, water, equipments. 22
  • 23. Transmission of Disease (A) Contact transmission • Direct contact – direct transmission between the source & a susceptible host. E.g. touching, kissing, sexual intercourse - anthrax, AIDS. • Indirect contact - agent of disease is transmitted from its reservoir to a susceptible host by means of nonliving object (fomite). E.g. tissues, beddings, thermometer - hepatitis B, tetanus. • Droplet transmission – spread of microbes in droplet nuclei in short distance i.e. less than 1m. E.g. Coughing, sneezing - influenza, pneumonia. 23
  • 25. Disease Transmission (B) Vehicle Transmission – Transmission of disease by a medium, i.e. water, food, air. – Waterborne – pathogens are spread by contaminated water, poorly treated sewage. E.g. cholera, shigellosis. – Foodborne – pathogens are spread by food improperly cooked, poorly refrigerated, are prepared under unsanitary conditions. E.g. food poisoning, tapeworm infections. – Airborne – pathogens are spread by dust particles that travel in air more than 1m in distance. 25
  • 26. (C) Vectors – Animals that carry pathogens from one host to another, E.g. arthropod & other insects. – Mechanical transmission – passive transport of the pathogens on the body parts of the vectors that makes contact with host’s food. – Biological transmission – active process in which vectors bite an infected person and pathogens multiply in vector and spreads to another host after biting the host. 26 Disease Transmission
  • 27. Nosocomial Infections • Any infection that is acquired during the course of stay in a hospital, nursing home or other healthcare facilities. • About 5-15% of all hospitalised patients. • Normal microbiota can be a causative agent. • Opportunistic pathogens, drug-resistant gram negative & gram positive bacteria. • Patients with burns, surgical wounds & suppressed immune system. • Transmitted by direct contact & fomites. • Control by aseptic techniques, proper cleaning, storage, handling of equipments & supplies by hospital staff. 27
  • 28. Factors of contribution to nosocomial infections 28
  • 29. Modes of disease transmission in a hospital 29
  • 30. Isolation of Clinical Samples • Important to diagnose a disease. • Samples to be taken aseptically. • Container with sample should be labeled – patient’s name, room number, date, time, medications taken. • Must be sent to lab immediately. • Cultured on selective or differential media to isolate & identify any abnormal microbes. 30
  • 31. Isolation of Clinical Samples • Precautions are to be taken by all healthcare workers, including students. (Refer to Appendix B of text book). • Instructions for taking clinical samples are specific from one culture to another of different types. • Wound/ Abscess, Ear, Eye, Blood, Urine, Faecal, Sputum. 31
  • 32. Antibiotic Sensitivity Testing AST Disc Diffusion Method Kirby- Bauer disc diffusion Stokes disc diffusion Dilution Method Broth dilution Agar dilution
  • 33. (A) Medium used • Mueller-Hinton agar or broth for aerobic & facultative anaerobic isolates. • Petri dish of size 100 mm in diameter & depth of 4 mm (25 mL). • pH of medium btw 7.2 and 7.4. 33
  • 34. (B) Inoculum • An overnight incubation is ideal. • Then about 5-10 colonies are removed and cultured in a broth culture for 4-6 hrs (log phase). • Density of organism should be appx. 108 cfu/ml. • Inoculum of a standard strain should be prepared as control. 34
  • 35. (C) Antibiotic discs • 6mm in diameter commercial discs with accurate antibiotic concentration. • Discs prepared in the lab should contain proper diluted antibiotic solution. • Discs and discs dispensers should be stored in a sealed container at -20°C (long term) or <8°C (immediate use). • About 7 discs on a 100 mm plate. 35
  • 36. Kirby-Bauer Disk Diffusion Method • An agar plate is swabbed 3 times with an inoculum suspension. • Lid is replaced and allowed to dry for 15 mins. • Antibiotic discs are applied using sterile forceps/ multidisc dispenser. • Distance between one disc & another should not be closer than 24 mm centre to centre. • Plates are incubated at 35-37°C, 16-18 hrs. 36
  • 37. Stokes Disc Diffusion method • Plate is divided into 3 parts: test organism in the centre, control on the upper and lower thirds OR control in the centre, test organisms in the other 2 parts. • An uninoculated gap of 2-3 mm to separate the test & control area. • A maximum of 6 antibiotic discs on a 100 mm plate. • Incubated at 35-37°C, 16-18 hrs. 37
  • 38. The principle of antibiotic diffusion in agar • As soon as antibiotic impregnated disc comes in contact with moist agar surface, it absorbs moisture from agar. • Antibiotic diffuses into the surrounding medium. • As the distance from disc increases, there’s a logarithmic reduction in antibiotic concentration. • Reference strains should be tested for each new batch using S. aureus, E. coli & P. aeruginosa. 38
  • 39. Interpretation of inhibition zones Eg. Chloramphenicol Resistant Intermediate Susceptible ≤ 12 mm 13-17 mm ≥ 18 mm 39
  • 40. Minimum Inhibitory Concentrations (MIC) • Is the least amount of antimicrobial agent that inhibits visible growth of an organism after overnight incubation. 40 Minimum Bactericidal Concentrations (MBC) Is the least amount of antimicrobial agent that prevents bacterial growth after subculture of the organism into an antimicrobial-free medium.
  • 41. MIC • MIC can be determined in liquid or solid medium (Mueller-Hinton). • Antimicrobial agents of concentrations 0.25-125 µg/ml or other test concentrations are used. • For broth dilution, final inoculum should be 105 cfu/ml & on agar about 1-2 µL is used. • It should deliver about 104 cfu/spot. • Incubation is at 35-37°C, 16-18 hrs (depends on microbe). • The lowest concentration at which there is no visible growth is the MIC. 41
  • 42. MBC • Subculture is done from the tubes to a NB free from antimicrobial agents. • Incubated at 35-37°C, 16-18 hrs (depends on microbes). • Growth is examined. • The tube that contains the lowest concentration that fails to yield growth on subculture is the MBC. 42