1. Laboratory Methods For Identification Of Bacteria

2. 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

4. The hanging Drop Method

5. Commonly used stains 1- Simple stains 2- Differential 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)

6. Results Difference Gram (+) Purple Gram (-) Red
due to structure of cell wall
Gram (+) Thick cell wall
Gram (-) Thin cell wall
A Gram stain of mixed Staphylococcus aureus

7. Gram’s Stain

8. 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)

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

10. examine under oil immersion lens
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

11. Result Acid Fast organism Non 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)

12. 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

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)

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

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

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).

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.

19. 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

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.

21. 3-Differential media Differential media or indicator media Indicators
distinguish one microorganism type from another growing on the same media
Indicators
neutral red
phenol red
eosin Y
methylene blue

22. Examples of differential media include Eosin methylene blue (EMB)
differential for lactose and sucrose fermentation
E. coli on EMB agar

23. MacConkey (MCK) differential for lactose fermentation
A MacConkey agar plate with an active bacterial culture

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

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)

26. 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

27. Lofflers serum media Horse serum + glucose in a ratio 3:1
It is used for cultivation of Corynebacterium diphtheriae

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

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

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

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

33. Compounds that are still isolated from living organisms
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

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

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

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

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

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

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.

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.

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 hours incubation period is known as minimal inhibitory concentration (MIC)

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

43. Minimum Inhibitory Concentration