The document discusses various microbiology techniques for culturing microbes including inoculation, isolation, incubation, inspection, and identification. It describes how to produce pure cultures through methods like streak plating and describes different types of culture media including solid, liquid, enriched, selective, and differential media. The goals are to transfer microbes to produce isolated colonies, grow them under proper conditions, observe characteristics, and identify organisms through comparing data.

1. Microbiology
Culturing Microbes
Lab 4

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

20. Blood agar
• Rich with nutrients
• Differential (can see hemolysis)

22. Urease medium

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

27. Isolation: Producing CFU
• Plating methods

28. Isolation:
Streak Plates: not suitable for plate counts

29. Smear Plate
Rotation of glass wand spread microbes
across entire plate

31. 3. Incubation:
• Allow organisms to grow under the optimal conditions
• Temperature regulation
• With or without oxygen
• CO2%, etc.

32. Incubation:
• Allow organisms to
grow under the optimal
conditions
• Temperature, with or
without oxygen etc.
• Candle jar reduces
oxygen

33. 4. Inspection:
• Observation, description
• Colony Morphology, Microscopic examination (grams stain)
• Systematic recording of “DATA”

34. Inspection
Gram (+) bacilli Gram (-) bacilli

35. Acid fast, and capsule

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

39. Today’s Lab

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