1. Identification technique Bacterial Growth curve Bacterial reproduction

2. Staining stain is a salt with a negative and positive charged ion
color portion is a positive charge and will be attracted to the negative charged microbial cell
Simple staining
Differential staining
Enterobacter

3. Staining stain increases contrast between specimen and background
most microorganisms are colorless and difficult to see
Unstained Halophile
Stained Halophile

4. Staining simple stains positive staining
cells are dark against light background
makes cell shapes and arrangements visible
yeast stained with methylene blue stain

5. Staining negative staining
clear microorganisms against a dark background
better view of microbial shape due to lack of distortion
stain is not taken up by the microorganism
very useful for microorganisms with capsules
negatively stained mixed bacteria culture

6. Differential Staining
multiple stains
differentiate bacteria according to their reaction to the staining process
differentiation is based on differences in cell structure
Gram stain
acid fast stain

7. Differential Staining
Gram Stain
most widely used procedure
process uses a stain, wash, counterstain

8. Differential Staining
Gram Stain
Gram positive bacteria retain the purple stain
Gram negative bacteria appear pink after the counterstain
1.Primary stain
2.Complexing agent
3.Decolorizer
4.Secondary stain
Gram Gram +

9. Primary Stain – Methylene blue
Complexing agent – Grma’s iodine
Decolourizer – Acid Alcohol
Secondary Stain – Saffranine

10. Gram’s Staining
Smear
Methylene blue
Water wash
Gram’s iodine
(Timing)

11. Acid Alcohol
Water Wash
Saffranin
Observe under 10X
Observe under 45X
Observe under 100X

12. Observed Under 10X
Gram +Ve
Gram -Ve

13. Observed Under 45X
Gram +Ve
Gram -Ve

14. Observed under 100X
Gram +Ve
Gram -Ve

15. Mechanism of Action of Gram staining

16. After adding methylene blue After adding Grams Iodine
Gram +Ve
Gram -Ve
Less lipid content
more lipid content
After adding methylene blue
After adding Grams Iodine

17. lipid - Methylene blue complex due to presence of Mg.Ribo nucleate
No Complex due to Absence of Mg.Ribo Nucleate
After adding Acid Alcohol

18. After adding Saffranin
Small size pores
Big size pores
After adding Saffranin

19. After adding Saffranin Saffranin enters through big pores
Methylene blue complex not removed
Blue color cocci shape cells observed G+Ve
Pink color rod shape cells observed G-Ve

20. Observed under 100X
Gram +Ve
Gram -Ve

21. Differential Staining
acid-fast staining
waxy chemical in the cell wall allows the bacteria to retain the stain
Cryptosporidum muris

22. Differential Staining
acid-fast staining
makes Mycobacterium ( causative agent for tuberculosis) easy to recognize in sputum,
sputum sample containing
M. tuberculosis

23. Special Stains stains that color only certain parts of bacteria
endospore staining
reveals the presence of endospores
produced by few bacteria
Clostridium and Bacillus
A is the microorganism, B indicates the endospore

24. Special Stains flagella staining
adheres to an enlarges the flagella for ease of observation
flagella

25. Bacterial growth curve
Bacteria grow in population called Culture
Binary fission result in “geometric progression in number” which is termed exponential growth
1-> 2-> 4-> 8->16 -> 2n….
Generation time (doubling time):time needed for division

26. Growth curve

27. 4 Phases of growth curve Lag Phase Log Phase Stationary Phase
Decline Phase

28. Bacterial Growth & Metabolism
Batch or Limited Growth Curve
b/c bacteria reproduce by Binary Fission, the # of cells incr’s exponentially w/ time (exponential or log phase of growth)
1 cell becomes 2, 2 become 4, 4 become 8, 8 become 16, etc.
1 batch; just a limited supply of nutrients available
System is closed
No removal of waste products
No monitoring of pH
No aeration
No exogenous nutritional supplement

29. Bacterial Growth & Metabolism
4 major phases of growth
Lag Phase (NO growth)
Period of cell adjustment / preparation
Synthesis of components needed for subsequent growth
Cell mass & cell size incr. in readiness for division
Growth rate = 0; NO incr. in cell #
Log Phase (Balanced or Exponential Growth)
Balanced growth – all measurable components of the cell increase at the SAME rate (i.e., PRO, DNA, RNA, mass); doubling of cells (12481632, etc.)
Exponential growth – constant growth; cells divide at constant rate
Both cell mass & cell # incr. in coordinate manner
GROWTH RATE IS GREATEST
GENERATION TIME IS SHORTEST
Growth rate - # of generations of new cell formed in one hour
Generation time – the time to go from one generation to the next
N = # of cells, n = # of generations, I = an initial or early count, f = final or later count

30. Bacterial Growth & Metabolism
4 major phases of growth
Stationary Phase
Entry into stationary phase d/t
Accumulation of waste products
Exhaustion of nutrients
Change in pH
Decrease in O2 levels
NO increase in # of viable cells
# of viable cells = # of dying cells
The overall total of living & dead cells is HIGHER in stationary phase vs. log phase
Most resistant to environmental changes (heat, cold, drying, radiation, etc)
*** Stationary Phase w/ largest Total # of cells (both living & dead); Log phase w/ largest # of Viable cells.

31. Bacterial Growth & Metabolism
4 major Phases of Growth
Decline or Death Phase
Growth rate decreases
Death rate increases
Limited amount of cellular growth; availability of nutrients exhausted
Dead cells provide nutrients for remaining viable cells (thus, there are some living cells in this phase)
Clinical Note: Log Phase is MOST effective stage for human infection; bacteria are growing the fastest & generation time = shortest

32. Reproduction of bacteria
Asexual - Binary fission
Sexual - Conjugation
- Transduction
- Transformation

33. Bacteria Reproduction
Bacteria reproduce asexually by BINARY FISSION.
In ideal environments, bacteria can divide every 20 minutes!

34. Bacteria Reproduction
Bacteria may swap genes and therefore alter their genetic make up
What is selective advantage for obtaining new genes?
CONJUGATION
Obtain plasmid or swap plasmid with another cell thru sex pilus
TRANSFORMATION
Pick up naked DNA from environment
TRANSDUCTION
Obtain new genes from viruses

35. Bacteria Reproduction
Bacteria reproduce by the process of binary fission.
Binary fission involves the copying of the DNA and the splitting into two new cells.

36. Bacteria Reproduction
Under optimum conditions bacteria can reproduce every 20 minutes.
Bacteria reproduction is controlled by various factors including : temperature and food availability.

37. Conjugation
Some bacteria can reproduce sexually in a process called conjugation.
In conjugation one bacteria is able to transfer its DNA into another bacteria by means of a pilus (pili)