2. The Five “I’s
1. Inoculation: Method of transferring organisms
used to produce a pure culture
2. Isolation: Selection of a single type of colony
on media (CFU) with one single kind of microbe
growing (pure culture/isolated colony)
3. Incubation: Method of growing microbes under
proper conditions
4. Inspection: Observation of characteristics, data
and results
5. Identification: use of data, comparison
correlation, to ID organism to exact species
3. 1. Inoculation: Producing a pure
cultures
• Colony
– macroscopically visible collection of bacteria
originating from a single bacterial cell.
• Colony Forming Units (CFU)
– All the genetically related progeny of a single
cultured organism or cell growing as colony.
• Pure cultures
– a single organism and its reproduced progeny
4. 1. Inoculation:
• Introduce bacteria into a growth medium using “aseptic
technique” to prevent contamination.
• Tools: Bunsen burner, loop. Needle, etc.
5. Inoculation:
Agar Gel
• Frau Hesse
• Used for preparing solid medium
• Obtained from seaweeds.
• No nutritive value
• Not affected by the growth of the bacteria.
• Melts at 98oC & sets at 42oC
• 2% agar is employed in solid medium
6. 2. Isolation: Producing CFU
• Isolation:
– Growth media/medium:
• Liquid/gel designed to support the growth of microbes, cells or small
plants
– General :
• NA (nutrient agar), TSA (Trypticase soy agar)
– Differential: Have dyes, salts, inhibiting agents
• Mac (selectively isolate Gram- ve & enteric bacilli and differentiate
them based on lactose fermentation.
• EMB (Eosin methylene blue is a selective stain for gram-negative
bacteria)
• SS (Salmonella-Shigella , is a selective and differential medium . It is
used for the isolation, cultivation and differentiation of gram-negative
7. Pure Cultures
• Pure culture: culture of bacteria in which the
organisms are all genetically identical
– All of the organisms are the same species and
contain the exact same DNA (clones)
– Usually they are all derived from the same original
cell
• Mixed Culture – culture of bacteria in which
the organisms are not genetically identical
– The organisms are different species and/or
contain different DNA
8. • Pure cultures are necessary to ensure the
integrity of your results
– Different bacteria have different properties and
respond differently to chemicals and other
manipulations
– If your culture is not pure, how can you be sure
your results are accurate?
• Contamination by another type of
microorganism can ruin all of your work
Why are pure cultures important?
9. What is an “isolated colony?”
• Colony – aka Colony Forming Unit (CFU)
– Small culture of bacteria, on solid media
• all derived from the same original bacterial cell
– They are all genetically identical (therefore, this is
a type of pure culture)
• Isolated colony
– CFU not touching or questionably close to any
other CFU on the plate or slant
– Obtaining isolated CFUs from a culture (mixed or
pure) is the point of the 3 techniques
10. Types of culture media
1. Based on consistency
a) solid medium
b) liquid medium
c) semi solid medium
11. Solid media – contains 2% agar
• Colony morphology, pigmentation, hemolysis can
be observed.
• Eg: Nutrient agar, Blood agar
Liquid media – no agar.
• For inoculum preparation, Blood culture, for the
enrichment of pathogens from a mixture.
• Eg: Nutrient broth, Selenite broth
Semi solid medium – 0.5% agar.
• Eg: Motility medium
12. Types of culture media
II. Based on the constituents/ ingredients
a) simple medium
b) complex medium
c) synthetic or defined medium
d) Special media
Special types of media:
– Enriched - Enrichment
– Selective - Indicator - Differential
– Sugar - Transport - Biochemical reactions
13. Simple media / basal media
• Eg: Nutrient Broth, Nutrient Agar, Tryptic
Soy Agar
• NB consists of peptone, meat extract, NaCl,
• NB + 2% agar = Nutrient Agar
14. Complex media
• Media other than basal media
• They have added ingredients
• Provide special nutrients
Synthetic or defined media
• Media prepared from pure chemical
substances and its exact composition is
known
• Eg: peptone water – 1% peptone + 0.5%
NaCl in water
15. Enriched media
• Substances are added to the basal medium
– blood, serum, egg
– For bacteria with strict nutritional needs
• Blood agar (L)
• Chocolate agar (R)
16. Enrichment media
• Liquid media used to enrich
pathogens in mixed cultures
• Media is incorporated with
inhibitory substances to
suppress unwanted organisms
(e.g normal flora)
• Selenite F Broth
– Isolation of Salmonella, Shigella
• Alkaline Peptone Water
– Isolation of Vibrio cholerae
17. Selective media
• Inhibitory substances added to
solid media
• MacConkey
– For gram -ve bacteria
– Has crystal violet and bile salts to
inhibit gram +ve organisms
– Lactose fermentation is differential
• TCBS (Thiosulfate-citrate-bile salts-sucrose )
– For Vibrios
– Has sodium thiosulfate and sodium
citrate to inhibit Enterobacteriaceae
– Sorbitol fermentation is differential
18. Selective media
• LJ (Lowenstein Jensen) medium
– M. tuberculosis
– Malachite green, eggs, etc
• Potassium tellurite medium
– Diphtheria bacilli
19. Indicator media
• Contain an indicator which changes its
color when a bacterium grows in them.
– Blood agar
– MacConkey
– Christensen’s urea
23. Differential:
• Mac, EMB, SS
• These have dyes, salts, inhibiting agents
– Visual differences on plates
24. Differential media
• Has substances which
permit differentiation.
– Mac Conkey’s medium
– Peptone
– Lactose
– Agar
– Neutral red
– Taurocholate (bile salts)
• Distinguish between:
– Lactose fermenters
• Pink colonies (E. coli)
– Non lactose fermenters
• Colorless (Salmonella)
25. Sugar media
• Media containing
fermentable substance
– Glucose
– Arabinose
– Lactose
– Starch etc
• Contains 1% sugar,
peptone, phenol red
• Durham tube
– small tube for gas detection
26. III.Based on Oxygen requirement
- Aerobic media
- Anaerobic media
Types of culture media
36. 5. Identification:
• Correlating data from all observations and
resources to ID organism to species
– Flow charts
– Bergey’s manual, etc.
– API (Analytical profile index)
37. 5. Identification:
• Correlating data/resources to
ID organism
– Gram (+)
– Cocci
– “Grape” clusters
– Golden yellow colonies,
– Catalase +
– Coagulase +
– Resistant to Methicillin (MRSA,
Methicillin-resistant Staphylococcus aureus )
• Staphylococcus aureus
40. HOW TO OBTAIN PURE CULTURES:
Methods of Bacterial Separation
Quadrant Streak Plate:
• Method to dilute inoculum and obtain well-
isolated and discrete colony forming units
• Spread Plate: Method of amounts of
organisms often for the purpose of counting,
using a glass rod
41. Today
• Describe 6 plates
– Two on simple media (TSA)
– Two on enriched media
(BAP)
– Two on selective media
(MAC)
– Note: form, elevation,
margin, color, hemolysis
(BAP only), lactose
reaction (MAC only)
42. Today
• Prepare 3 Streak plates
– One on simple media
– One on enriched media
– One on selective media
