 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
The hanging Drop Method
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)
 Results
 Gram (+)
 Gram (-)
Purple
Red
 Difference
 due to structure of cell wall


Gram (+)
Gram (-)
Thick cell wall
Thin cell wall
A Gram stain of mixed Staphylococcus aureus
Gram’s Stain
Ziehl–Neelsen stain
 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)
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
 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
Result
 Acid Fast organism
 Red as Mycobacterium tuberculosis
 Non Acid Fast organism
 Blue as Enterobacteriaceae family
A. Non Acid-fast bacteria
B. Acid-fast bacteria
Mycobacterium tuberculosis
(stained red) in tissue (blue)
Special stains
 Capsule stain and Flagella stain
Pseudomonas fluorescens cultured on
nutrient agar, stained using
the Presque Isle flagella stain
Encapsulated Bacillus sp. stained using
Maneval's capsule staining method
(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)
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
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
 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).
 Blood-free, charcoal-based selective medium agar
(CSM)
 isolation of Campylobacter sp.
Blood-free, charcoal-based
selective medium agar (CSM) for
isolation of Campylobacter.
 Löwenstein–Jensen medium
 enriched selective media for T.B.

Distinctive clusters of colorless
Mycobacterium tuberculosis
Löwenstein-Jensen medium used for growing
M. tuberculosis in a McCartney bottle
 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.
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
 Examples of differential media include
 Eosin methylene blue (EMB)
 differential for lactose and sucrose fermentation
E. coli on EMB agar
 MacConkey (MCK)
 differential for lactose fermentation
A MacConkey agar plate with an active bacterial culture
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
 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)
 Chocolate agar (heated blood agar)
 enriched with heat-treated blood (40-45°C).
Comparison of two culture media types used to grow Neisseria gonorrhoeae bacteria
 Lofflers serum media
 Horse serum + glucose in a ratio 3:1
 It is used for cultivation of Corynebacterium diphtheriae
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
 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
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
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
 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
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
 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
 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
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
 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
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.
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.
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)
 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
Minimum Inhibitory Concentration