Introduction to Medical

Microbiology
DM SUKRAMA
DEPT.MICROBIOLOGY FACULTY OF
MEDICINE UDAYANA UNIVERSITY

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 Why study Microbiology ??
Microbiology as a BASIC Science
Bacteria and yeast are useful in studying molecular biology, biochemistry
and genetics
--reproduce rapidly
--are genetically (DNA) and biochemically more simple than
higher order organisms
--working with bacteria and yeast for understanding life processes
has no ethical ramifications

Microbiology as an APPLIED Science

Medicine—Vaccine development, production of antibiotics,
production of important biological enzymes (insulin)
Industry—Production of beer, wine, cheeses and yogurt
Agriculture—maintenance of soil fertility/digestion in cattle
Ecology—Bioremediation—microorganisms that degrade
toxic waste materials

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 Major Differences between prokaryotic
and eukaryotic micro-organisms
Prokaryotes Eukaryotes
1. Nonmembrane bound 1. Membrane bound nucleus
nucleiod region containing DNA
2. DNA-one circular molecule 2. DNA-linear molecules arranged
one chromosome to form several chromosomes
3. Haploid-One copy of a gene 3. Diploid-Two copies of a gene
4. Plasma membrane does not 4. Plasma membrane contains
contain sterols sterols
5. Reproduction—simple binary 5. Reproduction—meiosis and
fission mitosis
6. Presence of membrane bound
organelles such as chloroplasts
and mitochondria

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 Three Domain System
Eucarya
Eubacteria Archaea

Protista
Methanogens Animalia
Gram positives Halophiles Fungi
Gram negatives Thermoacidophiles Plantae
Spirochetes
•Circular DNA chromosome • Linear DNA chromosomes
Cyanobacteria •Prokaryotic cell type • Eukaryotic cell type
•Circular DNA chromosome •No peptidoglycan in cell walls• Cell walls variable if
•Prokaryotic cell type present
•Branched chain phospholipids
•Peptidoglycan cell walls •Antibiotic insensitive • Straight chain
•Straight chain phospholipids •Methionine for first aa phospholipids
•Antibiotic sensitive • Antibiotic insensitive
•F-methionine for first aa • Methionine for first aa
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 Aims
• What is medical microbiology?
• Why is it relevant?
• Some important concepts.
• Basic classification of organisms.
• Classifying bacteria.

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What is Medical Microbiology?
“the study of microorganisms (including
bacteria, viruses, fungi and parasites)
which are of medical importance and are
capable of causing diseases in human
beings”

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 What is Medical Microbiology?
What organisms cause infection?
How they cause infection.
How to treat them.
How to prevent infection.

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• Medical microbiology is the study of causative
agents of infectious diseases of humans and
their reactions to such infections. In other
words it deals with etiology, pathogenesis,
laboratory diagnosis, specific treatment and
control of infection (immunization).

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 Why is it Important?
• Infection is one of the most important
causes of mortality and morbidity in the
population.
• Approximately 30% of hospital patients are
on antibiotics at any one time
• 1 in 10 patients acquires an infection
whilst in hospital.

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 A Few Concepts
• Normal Flora
• Contamination
• Colonisation
• Infection

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 Normal Flora
• Human beings are not microbiologically
sterile.
• We are ALL covered with bacteria, fungi
and some parasites.
• Skin, nose, mouth, gastrointestinal tract …
• ~109 bacteria per gram of faeces
• Each person carries more non-human
cells on their body than their own
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 Normal Flora
Why?
• Fulfil a range of useful functions (symbiotic in
some cases).
• Prevent other, pathogenic bacteria from gaining
a foothold:
• by taking up space
• competing for nutrients.
• In the gut they aid digestion & produce essential
vitamins (folic acid & vitamin K).
• Can cause disease IF they get into the wrong
site- e.g. perforated appendix. 12
 Contamination
• Presence of an organism in a culture that
was not in the sample when taken.
• e.g. a culture of blood contaminated with
an organism from the skin
• sample contaminated in the lab

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 Colonisation
• Presence of an organism at a site but not
causing a tissue reaction (inflammation),
symptoms or disease.
• Could be normal flora
• Could be abnormal flora-such as after the
patient has received antibiotics.

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 Infection
• where organisms invade a body site and
their multiplication initiates a tissue
reaction producing symptoms/disease.

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 Three Domain System
Eucarya
Eubacteria Archaea

Protista
Methanogens Animalia
Gram positives Halophiles Fungi
Gram negatives Thermoacidophiles Plantae
Spirochetes
•Circular DNA chromosome • Linear DNA chromosomes
Cyanobacteria •Prokaryotic cell type • Eukaryotic cell type
•Circular DNA chromosome •No peptidoglycan in cell walls• Cell walls variable if
•Prokaryotic cell type present
•Branched chain phospholipids
•Peptidoglycan cell walls •Antibiotic insensitive • Straight chain
•Straight chain phospholipids •Methionine for first aa phospholipids
•Antibiotic sensitive • Antibiotic insensitive
•F-methionine for first aa • Methionine for first aa
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 Classification of Organisms
• All living organisms are classified into:
• Kingdom
• Phyllum (family)
• Genus
• Species
• Organisms that can cause disease are
many and varied and include:
• Viruses
• Bacteria
• Fungi
• Parasites
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 Relevance of Classification
• Different:
– Diseases
– Modes of transmission
– Treatment-e.g. antibiotics don’t cure viral
infections

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 Viruses
Small (50-300nm)
Unable to replicate
independently
Invade host cells and use
their cellular machinery to
replicate
Influenza, Chickenpox
(varicella), Herpes,
Rhinovirus, HIV/AIDS
Often difficult to treat 19
• Obligate
intracellular
parasites have one
type of nucleic acid,
DNA or RNA
surrounded by a
protein coat called
capsid.

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 Bacteria
• 500-800nm
• Capable of
independent
replication
• Cause of most
infections seen in
hospital
• Pneumonia, bacterial
meningitis, cellulitis,
UTI…
• Many different species
• Treated with
antibiotics 21
 Morphology of Bacteria
• Bacteria are intracellular free-living
organisms having both DNA and RNA. Their
biological properties and predominant
reproduction by binary fission relates them to
prokaryotes.
• Spherical (cocci)
• Rod-shaped
(bacteria, bacilli,
and clostridia)
• Spiral-shaped
(vibriones, spirilla, spirochaetes) 22
 Spherical
(cocci)
bacteria
1.Micrococci
2. Diplococci
3. Streptococci
4. Staphylococci
5. Tetracocci
6. Sarcine
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 Fungi
• Complex, large organisms
• Eukaryotes (as are
humans!)
• Divided into yeasts &
moulds
• Cause a range of
diseases e.g.:
– Thrush
– Athletes foot
– Invasive & allergic
aspergillosis
• Many diseases are
opportunistic.

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 Classifying Bacteria
Why bother?
Different bacteria:
• cause different diseases
• are susceptible/resistant to different
antibiotics
• some bacteria are common normal flora
whilst other closely related species are
pathogens
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 Classifying Bacteria
How?
• 1st into broad groups based on
microscopic appearance
• Then divided into species based on a
range of different properties-often
biochemical reactions e.g. some may be
able to metabolise a sugar that others
cannot.

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 Gram Stain
Method of differentiating bacteria.
Can be either Gram +ve or Gram –ve
depending on how they appear with the
stain.
Can then be further grouped based on
shape (rod=long thin or coccus=round).
Thus we end up with 4 combinations:
G+ rod, G+ coccus, G- rod, G- coccus
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BACTERIAL CELL

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Bacterial cell wall

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 Gram Stain
G+ve G-ve

• STAIN the slide with

crystal violet for 1-2
min.
• Flood slide with Gram's
iodine for 1-2 min.
• Decolourise by washing
the slide briefly with
acetone (2-3 seconds).
• Stain with safranin
counterstain for 2 min.
• View under microscope 30
 Gram Stain
Gives an initial idea of the possible identity
of the organism.
Can be done without growing the organism
(i.e. rapid result)
Thus can be done on pus, joint fluid,
sputum, CSF
1st result available on blood cultures

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 Gram Stain
Relevance of Gram reaction.
• Gram +ve and gram –ve organisms ae
susceptible to different groups of
antibiotics.
• Cause different diseases
• Differ in their ability to survive in the
environment-cleaning, infection control,
outbreak management.
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 fuchsine

Gram
Staining
Technique
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GPC
• Clusters: usually
characteristic of
Staphylococcus spp.,
such as S. aureus

• Chain or pairs:
usually characteristic
of Streptococcus
spp., such as S.
pneumoniae 34
GPR
• Thick : usually
characteristic of
Clostridium spp., such as
C. perfringens, C. difficile,
C. tetani

• Thin: e.g. Listeria spp.

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GNC
• Diplococci: usually
characteristic of Neiseria spp.,
such as N. meningitidis or N.
gonorrhoea. Though In
addition, Moraxella spp. and
Acinetobacter spp.are often
diplococcal in morphology.

• Coccobacilli: usually
characteristic of Acinetobacter
spp., which can be either
Gram-positive or Gram-
negative, and is often called
Gram-variable.

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GNR

• Thin rods: usually

characteristic of
enterobacteriaceae
(coliforms), such as E.
Coli

• Coccobacilli: usually
characteristic of
Haemophilus spp., such
as H. influenzae

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GNR

• Curved: usually
characteristic of Vibrio
spp.or Campylobacter
spp., such as V.
cholerae, C. jejuni

• Thin needle
shape: usually
characteristic of
Fusobacterium spp.

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Medical Microbiologists
Medically qualified (mostly)
Don’t spend time looking down
microscopes…
Clinical liaison-
• interpretation of results
• advice re Abx
• appropriate testing
MDT’s (ICU, Haematology, Renal, GUM…)
Infection control
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Methods of laboratory diagnosis
1. Bacterioscopical
2. Bacteriological
3. Detection
sensitivity of
bacteria to
antibiotics
4. Serological
5. Biological
6. DNA-technology
test (PCR) 40
BACTERISCOPIC
METHOD

Neisseria meningitidis

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BACTERISCOPIC
METHOD

Neisseria
meningitidis

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Bacteriological
method

Neisseria meningitidis

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 Serologic method

Agglutination test

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Serologic method

Agglutination test

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Serologic method

Agglutination test
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Allergic method

Mantoux’s test

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Express-diagnosis

IFT
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 Bacteria Frequently Present as Normal Flora Occasionally
Causing Overt Disease
Organisms Usual Locale Infectious Disease Process
Staphylococcus Nose, skin In all areas of the body,
aureus nosocomial diseases, food
poisoning
Staphylococcus Skin, nose, vagina Endocarditis, nosocomial
epidermidis phlebitis, acne
Enterococci Feces Blood, wounds, urinary tract,
endocarditis
Viridans Saliva Endocarditis
streptococci
Peptostreptococ- Mouth, feces, Abscess formation, gangrene
cus sp vagina
Neisseria sp Throat, mouth, Meningitis
nose
Veillonella sp. Mouth, vagina Bacterial endocarditis, abscesses
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Lactobacillus sp. Mouth, feces, Bacterial endocarditis (rare),
vagina lung abscess (1 report)
Corynebacterium Nasopharynx, Bacterial endocarditis
sp. skin, vagina
Mycobacterium Prepuce, Suspected in some infectious
(not clitoris, lung, disease processes
Mycobacterium feces, tonsils,
tuberculosis) Food, skin
Clostridium sp. Feces, skin, Clostridia myositis, cellulitis,
environment, food poisoning
including food,
Vagina
Enterobacteriaceae Feces, vagina, Urinary tract, wounds,
mouth, urethra pneumonia, nosocomial scesses,
meningitis, blood, peritonitis,
enteritis, abscesses, etc .
Moraxella sp. Nose, genito- Conjunctivitis, etc
urinary tract
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Achromobacter sp. Nose, Meningitis, blood, urethritis,
genitourinary burns
tract, skin
Pseudomonas sp. Feces, skin Blood, burns, wounds, urinary
tract, respiratory tract,
meningitis
Alcaligenes Feces Blood, urinary tract, conjunctiva,
faecalis respiratory tract, meningitis
Haemophilus sp. Nasopharynx, Laryngotracheobronchitis,
conjunctiva, meningitis, pyarthrosis, conjuncti
vagina vitis, genitourinary tract
Fusobacterium sp. Mouth, saliva, Infected human bites, gangrene
feces
Bacteroides sp. Feces, mouth, Bactenal endocarditis, abscesses,
throat mixed infections

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 Examination of the blood

Bacteriemia, septicemia, septicopyemia

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 Examination of the blood

Bacteriemia, septicemia, septicopyemia

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 Examination of urina

Etiology of urethritis
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Major causes of urinary tract infections

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Pathogenesis of genital tract infections
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Major causes of vaginitis Major causes of cervicites

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CEREBROSPINAL FLUID

Etiology of meningitis
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RESPIRATORY TRACT AND MOUTH

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60
Transtracheal aspiration A pillow should be placed beneath the
neck to permit maximum extension or the neck. After cleansing
the skin, a 14 gauge needle is inserted into the trachea, and a
polyethylene tube is passed through the needle into the lung 1 he
needle is withdrawn, and the tube is connected to a syringe
containing 3 ml to 4 ml of physiologic saline. The saline is
injected into the lung and immediately with drawn for culture. 61
 ORGANISMS COMMONLY ISOLATED FROM RESPIRATORY
TRACT SAMPLES AND SPECIALISED PROCEDURES USED
FOR THEIR IDENTIFICATION
Organism Special Procedures
Streptococcus group Sensitive to commercially available bacitracin
A hemolytic disks, catalase negative, fluorescently-labelled
antibody or conglutination
Streptococcus Sensitive to optochin disks; lethal for mouse in 18
pneumoniae hours
Staphylococcus Vogel Johnson medium; ferments mannitol;
aureus (coagulase positive)
Haemophilus Streak blood plate and check for hematin and NAD
influenzae requirement
Neisseria Grow in Thayer Martin medium
meningitides
Bordetella pertussis Bordet-Gengou agar plates
Corynebacterium Loeffler's coagulated serum and potassium
diphtheriae tellurite plates 62
Skin infection 63
Infections in which anaerobes are the predominant pathogens or are
commonly present

Region Type of Infection

Head and neck Brain abscess, Otogenic meningitis extradural or
subdural, Empyema,Chronic otitis media,
Dental infection
Pleuropulmonary Pneumonia secondary to obstructive process,
Aspiration pneumonia, Lung ahscess,
Bronchiectasis, Thoracic empyema
Intraabdominal Liver abscess, Pylephlebitis , Peritonitis,
Appendicitis, Subphrenic abscess, Wound
infection after bowel surgery or trauma, Liver
abscess
Female genital Puerperal sepsis, Postabortal sepsis,
Endometritis, Tuboovarian abscess
Other Perirectal abscess, Gas forming cellulitis, Gas
gangrene, Breast abscess 64
Concentration of microbes in human digestive tract
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 Main group of bacteria in the gut
Anaerobic bacteria
Bacteroides (B. fragilis), Prevotella,
Veilonella, Lactobacillus (L.
acidophylus, L. brevis), Clostridia
(C. perfringens, C. tetani, C.
botulinum, C.
sporogenes), Peptococcus,
Peptostreptococcus, Actinomyces
Aerobic and facultativly
anaerobic bacteria
E. coli, Enterococcus, Citrobacter,
Klebsiella, Proteus, Providencia,
Pseudomonas, Alcaligenes,
Bacillus (B. subtilis, B. Brevis),
Lactobacillus, Enterococcus,
Corynebacteria, Fungi, Candida
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Questions?

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