Bacteria are either identified in A pathological specimen obtained from the patient (e.g. pus,...
If you can't read please download the document
Bacteria are either identified in A pathological specimen obtained from the patient (e.g. pus, sputum, urine, blood, stools, etc.) depending on the site
Bacteria are either identified in A pathological specimen
obtained from the patient (e.g. pus, sputum, urine, blood, stools,
etc.) depending on the site of infection After been grown on
artificial nutrient media Bacteria are then identified by
Microscopic Examination Examination of fresh samples used for
demonstration of bacterial motility using hanging drop method
Morphology and staining reactions of bacteria
Slide 3
Slide 4
The hanging Drop Method
Slide 5
Commonly used stains 1- Simple stains e.g. methylene blue 2-
Differential stains e.g. Gram stain Primary stain Methyl violet
(Crystal Violet)- Iodine mixture Decolourization Alcohol Counter
stain Diluted carbol-fuchsin stain (Safranin)
Slide 6
Results Gram (+)Purple Gram (-)Red Difference due to structure
of cell wall Gram (+) Thick cell wall Gram (-) Thin cell wall A
Gram stain of mixed Staphylococcus aureus
Slide 7
Grams Stain
Slide 8
Differential Stain - divides bacteria into 2 groups Acid Fast
Non Acid Fast Used to identify organisms in the Genera
Mycobacterium (high lipid and wax content in cell wall)
ZiehlNeelsen stain
Slide 9
Procedure Fix the smear of the specimen over the glass slide
either by heating or alcohol fixation Pour carbol fuschin over
smear heat gently until fumes appear do not overheat allow it to
stand for 5 minutes wash it off with water
Slide 10
Pour 20% sulphuric acid 5% sulfuric acid is used for destaining
Mycobacterium leprae instead of the 20% used for Mycobacterium
tuberculosis wait for one minute keep on repeating this step until
the slide appears light pink in color wash off with water Pour
methylene blue wait for two minutes again wash with water Allow it
to air dry examine under oil immersion lens
Slide 11
Result Acid Fast organism Red as Mycobacterium tuberculosis Non
Acid Fast organism Blue as Enterobacteriaceae family Mycobacterium
tuberculosis (stained red) in tissue (blue) A. Non Acid-fast
bacteria B. Acid-fast bacteria
Slide 12
Special stains Capsule stain and Flagella stain Encapsulated
Bacillus sp. stained using Maneval's capsule staining method
Pseudomonas fluorescens cultured on nutrient agar, stained using
the Presque Isle flagella stain
Slide 13
Slide 14
(II) Cultural Characters Bacteria need nutritive culture media
to multiply in vitro An undefined medium (also known as a basal or
complex medium). It is a medium that contains: 1- A carbon source
such as glucose for bacterial growth 2- Water 3- Various salts
needed for bacterial growth Defined media (also known as chemically
defined media or synthetic media)
Slide 15
Classification of Media Media can be classified into 1-Minimal
media ( simple medium) It contains the basic nutritive requirements
e.g. nutrient broths and agar media
Slide 16
2- Selective media Selective media are used for the growth of
only selective microbes It contains antibiotics, dye, or specific
chemicals inhibits the growth of most types of microbe stimulate
the isolation of one type
Slide 17
Mannitol salt agar (MSA) selective for Gram positive (+ve)
bacteria An MSA plate with Micrococcus sp. (1), Staphylococcus
epidermis (2) and S. aureus colonies (3).
Slide 18
Blood-free, charcoal-based selective medium agar (CSM)
isolation of Campylobacter sp. Blood-free, charcoal-based selective
medium agar (CSM) for isolation of Campylobacter.
Slide 19
LwensteinJensen medium enriched selective media for T.B.
Lwenstein-Jensen medium used for growing M. tuberculosis in a
McCartney bottle Distinctive clusters of colorless Mycobacterium
tuberculosis
Slide 20
TCBS agar (Thiosulfate-citrate-bile salts-sucrose agar)
selective for Vibrio cholerae due to alkaline pH Yellow coloured
(sucrose fermenting) colonies of Vibrio cholerae on TCBS agar.
Slide 21
3-Differential media Differential media or indicator media
distinguish one microorganism type from another growing on the same
media Indicators neutral red phenol red eosin Y methylene blue
Slide 22
Examples of differential media include Eosin methylene blue
(EMB) differential for lactose and sucrose fermentation E. coli on
EMB agar
Slide 23
MacConkey (MCK) differential for lactose fermentation A
MacConkey agar plate with an active bacterial culture
Slide 24
4- Enriched media Enriched media contain the nutrients required
to support the growth of a wide variety of organisms including some
of the more fastidious ones Blood agar Is an enriched medium in
which nutritionally rich whole blood supplements the basic
nutrients It contains 5-10% human or animal blood
Slide 25
It shows the type of haemolytic activity of bacteria (complete,
partial or non-haemolytic) Complete Haemolysis of RBCs (Beta
Haemolytic Streptococci) Partial Haemolysis of RBCs (Alpha
Haemolytic Streptococci)
Slide 26
Chocolate agar (heated blood agar) enriched with heat-treated
blood (40-45C). Comparison of two culture media types used to grow
Neisseria gonorrhoeae bacteria
Slide 27
Lofflers serum media Horse serum + glucose in a ratio 3:1 It is
used for cultivation of Corynebacterium diphtheriae
Slide 28
5- Transport media Transport medium is a simple organic medium
maintain the viability of all organisms in the specimen without
altering their concentration This type of medium mainly used for
temporary storage of specimens being transported to the laboratory
for cultivation
Slide 29
Examples of transport media include Thioglycollate broth for
strict anaerobes Thioglycollate broth medium is recommended to
isolate strict anaerobes should an anaerobic infection be
suspected
Slide 30
Slide 31
The colonial appearance on culture media Shape The colonies may
be small (pin-point) fimbriate, flat or convex Colour The colonies
may be colorless or bacteria produce endopigments which give the
colonies a characterestic colour Staph. aureus produce golden
yellow colonies Staph. albus produce white endopigment Staph.
citreus produce a lemon yellow endopigment The bacteria may produce
exopigments Pseudomonas aeruginosa produce a green exopigments in
the surrounding media
Slide 32
Antimicrobial Chemotherapy An antibacterial agent is a compound
or substance that kills or slows down the growth of bacteria
Antibiotic(s) has come to include a broader range of antimicrobial
compounds, including anti-fungal and other compounds It is produced
by microbes and is harmful to other microbes, except viruses
Slide 33
These include beta-lactam antibacterial penicillin (produced by
Penicillium notatum) cephalosporin Compounds that are still
isolated from living organisms Aminoglycosides Other
chemotherapeutic agents produced by chemical synthesis Sulfonamides
Quinolones
Slide 34
Classification of Antibiotics According to agent action
Antibacterial agents are divided into two broad groups based on
their biological effect on microorganisms bactericidal agents kill
bacteria bacteriostatic agents slow down or stall bacterial
growth
Slide 35
Bactericidal antibiotics Antibiotics that inhibit cell wall
synthesis Beta-lactam antibiotics penicillin derivatives, and
cephalosporins Aminoglycosidic antibiotics are usually considered
bactericidal although they may be bacteriostatic with some
organisms
Slide 36
Bacteriostatic antibiotics limit the growth of bacteria by
interfering with bacterial protein production DNA replication Or
other aspects of bacterial cellular metabolism This group includes
Tetracyclines Sulphonamides Trimethoprim Chloramphenicol
Macrolides
Slide 37
Antibiotic sensitivity test Antibiotic sensitivity is a term
used to describe the susceptibility of bacteria to antibiotics
Antibiotic susceptibility testing (AST) is usually carried out to
determine which antibiotic will be most successful in treating a
bacterial infection in vivo
Slide 38
Testing for antibiotic sensitivity is often done by the
Kirby-Bauer method ( Disc-diffusion method) Other methods to test
antimicrobial susceptibility include the E-test (also based on
antibiotic diffusion) Agar and Broth dilution methods for Minimum
Inhibitory Concentration determination
Slide 39
In Kirby-Bauer testing, white wafers containing antibiotics are
placed on a plate of bacteria. Circles of poor bacterial growth
surround some wafers indicating susceptibility to the
antibiotic.
Slide 40
This is most commonly used in the setting of medicine, where a
particular organism has been found to infect a patient, and the
doctor treating the patient is seeking guidance on what
concentration of antibiotic is suitable.
Slide 41
The Dilution Method Serial dilutions of antibiotics are
incorporated in agar containing or broth culture media The lowest
concentration of antibiotic that prevents visible growth after an
18-24 hours incubation period is known as minimal inhibitory
concentration (MIC)
Slide 42
The minimal bactericidal concentration (MBC) may be determined
in broth dilution tests by subculturing the containers that show no
growth on to antibiotic-free agar containing media The lowest
concentration of antibiotic that totally suppresses growth after
overnight incubation is known as MBC