1. Increase in number of cells, not cell size Populations Colonies
Microbial Growth
Increase in number of cells, not cell size
Populations
Colonies

2. Culture Media
Culture medium: Nutrients prepared for growth of microorganisms
Sterile: No living microbes
Inoculum: Introduction of microbes into medium
Culture: Microbes growing in/on culture medium

3. Agar Complex polysaccharide
Used as solidifying agent for culture media
Slants: large surface area for growth
Deeps: show oxygen requirements
Plates: short term use

5. Types of Media
Chemically defined media: exact chemical composition is known (autotrophic bacteria)
Complex media: chemical composition may vary and includes nutrients including extracts from yeast, meat or plants (heterotrophic bacteria)

8. Selective and Differential Media
Selective Media: suppress growth of unwanted bacteria and encourage growth of desired bacteria.
Differential Media: distinguish colonies of desired organism from others growing on the same plate

9. Macconkey Agar-
Selects for Gram negative
Differentiates between bacteria that ferment lactose
Mannitol Salt Agar-
Selects for Gram Positive
Differentiates between bacteria that ferment mannitol (sugar alcohol) turn mannitol to acid causes color change

10. (selects for gram-)
(lactose fermentation)

11. Nutrient Agar
Commonly used for routine cultivation of bacteria

12. Biosafety Levels 1: No special precautions
2: Lab coat, gloves, eye protection
3: Biosafety cabinets to prevent airborne transmission
4: Sealed, negative pressure
Exhaust air is filtered twice

13. Biosafety Level 4 (BSL-4) Laboratory

14. Live Cell Cultures
Viruses are too small to view without special equipment that is very expensive
In the lab, we innoculate live cell cultures with the viral specimen
We examine the culture tubes for patterns of cell destruction by the virus (cytopathic effect or CPE)

15. Selective Media
By inhibiting unwanted organisms with salts, dyes, or other chemicals, selective media allow growth of only the desired microbes.

16. Differential Media
Make it easy to distinguish colonies of different microbes.
pH is always
Monitored to
Ensure optimal
Growth conditions
(most grow best
At pH 7)

17. Enrichment Culture Encourages growth of desired microbe
Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria
Inoculate phenol-containing culture medium with the soil, and incubate
Transfer 1 ml to another flask of the phenol medium, and incubate
Only phenol-metabolizing bacteria will be growing

18. Obtaining Pure Cultures
A pure culture contains only one species or strain
A colony is a population of cells arising from a single cell or spore or from a group of attached cells
A colony is often called a colony-forming unit (CFU)
The streak plate method is used to isolate pure cultures

19. The Streak Plate Method
Each streak dilutes the amount of bacteria until individual colonies can be seen

20. Preserving Bacterial Cultures
Deep-freezing: –50° to –95°C
Lyophilization (freeze-drying): Frozen (–54° to –72°C) and dehydrated in a vacuum

21. Direct Methods of Measuring Cell Growth
A standard plate count
Filtration
Most probable number (MPN) method
Direct microscopic count

22. Serial Dilutions
In order to more accurately count the colonies on the plates, the original sample is often diluted to reduce the total number of bacteria into a more “countable” range
If the original 1 ml of sample has 10,000 bacteria, we would dilute it until it was around 100 bacteria that is a more countable number on a plate
That plate # would be multiplied by the dilution factor to obtain the total original count of bacteria

23. Serial Dilutions
The 1:100,000 dilution was plated, incubated, & then counted to obtain 18 bacteria
What is the original # of bacteria?
1,800,000/ml

24. Plate Counts
# of viable microbes and assumes that each bacterium grows into a single colony

25. Plate Counts
After incubation, count colonies on plates that have 25–250 colonies (CFUs)

26. Counting Bacteria by Membrane Filtration
bacteria are retained on the surface of a membrane filter and then transferred to a culture medium to grow and subsequently be counted.

27. Most Probable Number Multiple tube MPN test Count positive tubes
used for microbes that will grow in a liquid medium
it is a statistical estimation.

28. Most Probable Number
Compare with a statistical table.

29. Direct Microscopic Count
The microbes in a measured volume of a bacterial suspension are counted with the use of a specially designed slide.

30. Direct Microscopic Count

31. Estimating Bacterial Numbers by Indirect Methods
Spectrophotometer
Measuring the metabolic activity
Measuring dry weight

32. Turbidity
Spectrophotometer is used to determine turbidity by measuring the amount of light that passes through a suspension of cells.

33. Turbidity

34. Metabolic activity
estimating bacterial numbers is measuring the metabolic activity of the population
for example, acid production or oxygen consumption

35. Measuring Dry Weight
For filamentous organisms such as fungi, measuring dry weight is a convenient method of growth measurement.