C
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Purification of cultures
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Morphological and pure culture studies
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Biochemical tests

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Reason to purify cultures.
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To characterize an individual species.
To study the morphology and physiology of individual bacterial species
To study their biochemical behavior and response.
To purify, pure cultures techniques can be used.Method:
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Streak plate method
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Pour plate method
Spread plate method

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Need to have a control procedure to avoid contamination .
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Specimen collection
Preparation of media
Microbiological tecniques
Staining and reagents
Equipment used.

S
C
 Applied the sterile techniques
 Use correct media for transportation and stock.
 The transport media used to preserve and ensure the viability of bacteria during the transportation period
 Important! Label your specimen.
 Crucial for cerebrospinal fluid, blood culture and fecal specimens, etc.

6
U
 Bacteria are everywhere
 Media used for bacteria growth  welcoming for many bacteria
 We only want specific ones to grow  Sterile techniques
 Sterile remain sterile as long as doesn’t touch anything that isn’t sterile
 Also avoid prolonged exposure to air

7
A
T
:
These are various techniques that are used to minimize the introduction of microorganisms into media especially during transfer processes, such as :

 pouring of media into Petri dishes inoculation of cultures
These techniques include:
 cleaning the bench top work areas with disinfectant solution
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 washing hands before starting work other specific techniques that will be demonstrated in the lab.

8
S
:
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Wash your hands
Keep your bench clean
Wear gloves
Flame loop, neck of tube
Keep cap facing down
Work quickly and efficiently
Limit talking when opening cultures

P
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The media should be packed well to prevent from leakage and breaks , protected from moisture and sunlight and excessive heat
The expiry date should be noted and the instruction of storage should be followed
The mix bacterial colonies should be sub cultured until the culture are purified the bacterial colony characteristic should only derive from a single colony

10
Plate
C
Broth
Slant Deep

M

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Morphological studies:
- Sizes, shapes, cell arrangement, cell wall, surface adherents or appendages,flagella,pili,endospores,ribosomes.
- Macroscopic examintation
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Techniques used in the study:
- Microscopic examintion
- Staining techniques

M

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P
B
Divide into 3 groups:
Selective media
Differental media
Enrichment media

S
 Prepared by the addition of specific subtances to a culture medium that will permit growth of one bacteria while inhibiting the growth of others.
 Contain antimicrobial agents such as crytal violet,bile salts,sodium azide,antibiotic and e.t.c.
Salmonella-Shigella Agar- media contain bile salts
(inhibits many coliform bacteria).Produce colorless colonies (unable to ferment lactose)
Mannitol Salt Agar -Isolation of Staphylococci.
Bismuth Sulfite Agar-Isolation for Salmonella typhi .Reduces the sulfite to sulfide results in black colonies and with metallic sheen .

D
 The incorporation of certain chemicals into a medium may result in diagnostically useful growth or visible change in the medium after incubation.
Eosin Methylene Blue(EMB)Differentiate between lactse and non-lactose fermenters .
Mac Conkey Agar-contain crystal violet and bile salts.Use for selection of Enterobacteriaceae and related gram negative rods.
Hektoen Enteric Agar-High concenration of bile salts.Inhibit Gram positive bacteria and retards the growth of many coliform strains.

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M
C

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C

E
 These are routinely employed in a laboratory e.g. nutrient broth, nutrient agar, infusion broth,blood agar.
 They support the growth of fastidious bacteria.

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B

H
 Destruction of erythrocytes nd hemoglobin in medium.
 Can be divided into 3 categories:alpha hemolysis, beta hemolysis and gamma hemolysis
Alpha hemolysis-greenish to brownish discolouration around the colonies. ( Streptococous gordonii,Streptococcus pneumoniae)
Beta hemolysis-complete lysis of blood cell resulting in clearing effect around the growth of colony.( S.aureus
)
Gamma hemolysis-no change in the medium.( Enterococcus faecalis)

B
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Catalase test
Oxidase test
Coagulase test
Sugar fermentation test
MRVP test
Indole test
Citrate test
Motility test
H
2
S test
Litmus milk test

C
 Produce bubble just after attaching the bacteria to the reagent
 To differentiate staphylococci and streptococci

O
 Have 2 methods:Filter paper/Sterile swab
 To help identify Vibrio, Neisseria, Pasteurella and
Pseudomonas sp.
 Oxidase enzymes oxydize phenylenediamine .
 Deep purple colour on reagent paper

O

 To identify S.aureus
 The enzyme coagulase clots plasma
 Tube : fibrin clot
 Slide: clumping of bacterial cells
C

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 Glucose test
 Maltose test
 Sucrose test
 Lactose test
 Some will appear with gas production

V
-P
 To differentiate enterobacteria
 Organism ferments glucose with acetoin production.
Acetoin is oxidised to diacetyl which reacts with creatine.
 Brick red colour develop slowly
 Eg: E.coli (-)
 Klebsiella sp. (+)

M
R
 To differentiate
Enterobacteria.
 Detect the production of sufficient acid during fermentation of glucose in buffered medium to give a colour change of indicator
 Brick red medium

I
 Using Kovac reagent .
 To differentiate Gram negative rods, especially E.coli .
 Demonstrates the ability of certain bacteria to decompose amino acid tryptophan to indole which accumulates in the medium.
 Reddening of strip or medium

I

C
 Test the ability of organism to utilise citrate as a sole carbon source and ammonium salt for nitrogen.Result in alkalinization in the medium with colour change indicator.
 Use Koser’s liquid citrate medium.
 Differentiate Enterobacteria from other bacteria.
 Positive result : Blue and turbid medium

M

LITMUS TEST
 Medium consisting of LACTOSE,CASEIN and the pH indicator azolitmin.
 It is used to differentiate members within the genus Clostridium . It differentiates Enterobacteriaceae from other Gram-negative bacilli based on enterics' ability to reduce litmus.
 The skim milk provides nutients for growth. The protein is casein and the lactose is for fermentation.
 Azolitmin is purple between pH of 4.6 and 8.2. It turns pink when pH reaches 4.5 and blue at a pH of 8.3.
 Because of this, litmus milk can give quite unreliable results .
Thus, you would be advised to use litmus milk as a confirmatory test but not a definitive test (except as a last resort).

T
 Triple Sugar Iron medium is a differential medium that can distinguish between a number of Gram-negative enteric bacteria based on their physiological ability (or lack thereof) to:
 a. metabolize lactose and/or sucrose b. conduct fermentation to produce acid c. produce gas during fermentation d. generate H
2
S.

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 Culture collection centre.
ATCC American type culture Collection Centre
NCTCC National Collection of Type Culture
NCIM Natonal Collection of Industrial and Marine Bacterial
NCDO National Collection of Dairy Organism

T