Pharmaceutical Microbiology

1. Unit III
Dr. Pabba Shivakrishna
Nutritional requirements and
types of media and growth
conditions…..

2. Nutritional Requirements of Cells
• Every organism must find in its environment all of
the substances required for energy generation
and cellular biosynthesis.
• The chemicals and elements of this environment
that are utilized for bacterial growth are referred
to as nutrients or nutritional requirements.
• Many bacteria can be grown the laboratory in
culture media which are designed to provide all
the essential nutrients in solution for bacterial
growth.

3. The Major Elements
• At an elementary level, the nutritional
requirements of a bacterium such as E. coliare
revealed by the cell's elemental composition,
which consists of C, H, O, N, S. P, K, Mg, Fe, Ca,
Mn, and traces of Zn, Co, Cu, and Mo. These
elements are found in the form of water,
inorganic ions, small molecules, and
macromolecules which serve either a
structural or functional role in the cells.

4. The Requirements for Growth
PHYSICAL REQUIREMENTS
– Temperature
– pH
– Oxygen
– Hydrostatic Pressure
– Osmotic pressure
CHEMICAL REQUIREMENTS
(NUTRITIONAL FACTORS)
– Carbon
– Nitrogen, sulfur, and
phosphorous
– Trace elements
– Oxygen
– Organic growth
factor

5. 1) pH
• Optimum pH: the pH at which the microorganism
grows best (e.g. pH 7)
• Most bacteria grow between pH 6.5 and 7.5
• Molds and yeasts grow between pH 5 and 6
• According to their tolerance for acidity/alkalinity,
bacteria are classified as:
Acidophiles (acid-loving): grow best at pH 0.1-5.4
Neutrophiles: grow best at pH 5.4 to 8.0
Alkaliphiles (base-loving): grow best at pH 7.0-11.5
Physical Factors Required for Bacterial Growth

6. 2) Temperature
• According to their growth temperature range, bacteria can be
classified as:
Psychrophiles : grow best at 15-20oC
Psychrotrophs : grow between 0°C and 20–30°C
Mesophiles : grow best at 25-40oC
Thermophiles : grow best at 50-60oC
 Typical Growth Rates and Temperature
– Minimum growth temperature: lowest temp which species can
grow
– Optimum growth temperature: temp at which the species grow
best
– Maximum growth temperature: highest temp at which grow is
possible

8. Food Preservation Temperatures

9. 3) Oxygen
• Aerobes: require oxygen to grow
• Obligate aerobes: must have free oxygen for aerobic respiration (e.g.
Pseudomonas)
• Anaerobes: do not require oxygen to grow
• Obligate anaerobes: killed by free oxygen (e.g. Bacteroides)
• Microaerophiles: grow best in presence of small amount of free oxygen
• Capnophiles: carbon-dioxide loving organisms that thrive under
conditions of low oxygen
• Facultative anaerobes: carry on aerobic metabolism when oxygen is
present, but shift to anaerobic metabolism when oxygen is absent
• Aerotolerant anaerobes: can survive in the presence of oxygen but do
not use it in their metabolism
• Obligate: organism must have specified environmental condition
• Facultative: organism is able to adjust to and tolerate environmental
condition, but can also live in other conditions

10. Patterns of Oxygen Use

11. 5) Osmotic Pressure
• Environments that contain dissolved substances
exert osmotic pressure, and pressure can exceed
that exerted by dissolved substances in cells
• Hyperosmotic environments: cells lose water and
undergo plasmolysis (shrinking of cell)
• Hypoosmotic environment: cells gain water and
swell and burst

12. Plasmolysis

13. Halophiles
• Salt-loving organisms which require moderate to large
quantities of salt (sodium chloride)
• Membrane transport systems actively transport sodium
ions out of cells and concentrate potassium ions inside
• Why do halophiles require sodium?
1) Cells need sodium to maintain a high intracellular
potassium concentration for enzymatic function
2) Cells need sodium to maintain the integrity of their
cell walls

14. Responses to Salt

15. The Great Salt Lake in Utah

16. Chemical Requirement: Nutritional
Factors
1. Carbon sources
2. Nitrogen sources
3. Sulfur and phosphorus
4. Trace elements (e.g. copper, iron, zinc, and
cobalt)
5. Vitamins (e.g. folic acid, vitamin B-12,
vitamin K)

17. Chemical Requirements
• Carbon
– Structural organic molecules, energy source
– Chemoheterotrophs use organic carbon sources
– Autotrophs use CO2

18. Chemical Requirements
• Nitrogen
– In amino acids and proteins
– Most bacteria decompose proteins
– Some bacteria use NH4+ or NO3–
– A few bacteria use N2 in nitrogen fixation

19. Chemical Requirements
• Sulfur
– In amino acids, thiamine, and biotin
– Most bacteria decompose proteins
– Some bacteria use SO4
2– or H2S
• Phosphorus
– In DNA, RNA, ATP, and membranes
– PO4
3– is a source of phosphorus

20. Chemical Requirements
• Trace elements
– Inorganic elements (mineral) required in small
amounts
– Usually as enzyme cofactors
– Ex: iron, molybdenum, zinc
• Buffer
– To neutralize acids and maintain proper pH
– Peptones and amino acids or phosphate salts may
act as buffers

21. Organic Growth Factors
• Organic compounds obtained directly from
the environment
• Ex: Vitamins, amino acids, purines, and
pyrimidines

22. Preparation of Culture Media
• Culture medium: Nutrients prepared for
microbial growth
• Sterile: No living microbes
• Inoculum: Introduction of microbes into
medium
• Culture: Microbes growing in/on culture
medium

23. Agar
• Complex polysaccharide
• Used as solidifying agent for culture media in
Petri plates, slants, and deeps
• Generally not metabolized by microbes
• Liquefies at 100°C
• Solidifies at ~40°C

24. CULTURE MEDIA USED
IN MICROBIOLOGY

25. Definition, purpose/importance
History of culture media
Classification of culture media
Growth pattern of bacteria

26. Microbiological culture
Method of cultivating microbial
organisms by letting them reproduce in
predetermined culture media under
controlled laboratory conditions.

27. Louis Pasteur used simple broths made
up of urine or meat extracts. Robert Koch
realized the importance of solid media
and used potato pieces to grow bacteria.
It was on the suggestion of Fannie
Eilshemius, wife of Walther Hesse (who
was an assistant to Robert Koch) that
agar was used to solidify culture media.
History of culture medias

28. Before the use of agar, attempts were made to
use gelatin as solidifying agent. Gelatin had some
inherent problems….
It existed as liquid at normal incubating
temperatures (35-37oC)
Digested by certain bacteria
Continued….

29. Agar
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

30. During typical bacteria growth (growth cycle)
bacteria cell divide by binary fission and
their mass and number increase in an
exponential manners. Bacterial growth in
culture can be separated into at least four
distinct phases.
Bacterial Growth Curve

31. Bacterial Growth Curve

33. The Lag Phase
• Organisms do not increase significantly in number
• They are metabolically active
• Grow in size, synthesize enzymes, and incorporate
molecules from medium
• Produce large quantities of energy in the form of ATP

34. The Log Phase
• Organisms have adapted to a growth medium
• Growth occurs at an exponential (log) rate
• The organisms divide at their most rapid rate
• a regular, genetically determined interval
(generation time)

35. Synchronous growth: A
hypothetical situation in
which the number of cells in a
culture would increase in a
stair-step pattern, dividing
together at the same rate
Nonsynchronous growth: A
natural situation in which an
actual culture has cell
dividing at one rate and other
cells dividing at a slightly
slower rate

36. 1) Cell division decreases to a point that new cells
are produced at same rate as old cell die.
2) The number of live cells stays constant.
Decline (Death) Phase
1) Condition in the medium become less and less
supportive of cell division
2) Cell lose their ability to divide and thus die
3) Number of live cells decreases at a logarithmic
rate
Stationary Phase

37. Measuring Microbial Growth
Direct Methods
• Plate counts
• Filtration
• MPN
• Direct microscopic
count
Indirect Methods
• Turbidity
• Metabolic activity
• Dry weight

38. Types of culture media
I. Based on their consistency
a) solid medium
b) liquid medium
c) semi solid medium
II. Based on the constituents/ ingredients
a) simple medium
b) complex medium
c) synthetic or defined medium
d) Special media

39. Special media
Enriched media
Enrichment media
Selective media
Indicator media
Differential media
Transport media
III.Based on Oxygen requirement
- Aerobic media
- Anaerobic media

40. Solid media – contains 2% agar
Colony morphology, pigmentation, hemolysis can be
appreciated.
Eg: Nutrient agar, Blood agar
Liquid media – no agar.
For inoculum preparation, Blood culture, for the
isolation of pathogens from a mixture.
Eg: Nutrient broth
Semi solid medium – 0.5% agar.
Eg: SIM

42. Simple media / basal media
- Eg: NB, NA
- NB consists of peptone, yeast extract, NaCl,
- NB + 2% agar = Nutrient agar

43. 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

44. Enriched media
Substances like blood, serum, egg are added
to the basal medium.
Used to grow bacteria that are exacting in
their nutritional needs.
Eg: Blood agar, Chocolate agar

45. Blood agar Chocolate agar

46. Enrichment media
Liquid media used to isolate
pathogens from a mixed culture.
Media is incorporated with
inhibitory substances to suppress
the unwanted organism.
Eg:
Selenite F Broth – for the isolation of
Salmonella, Shigella
Alkaline Peptone Water – for Vibrio
cholerae

47. Selective media
The inhibitory substance is added to a solid
media.
Eg:
Mac Conkey’s medium for gram negative
bacteria
TCBS – for V.cholerae
LJ medium – M.tuberculosis
Wilson and Blair medium – S.typhi
Potassium tellurite medium – Diphtheria bacilli

48. TCBSMac Conkey’s medium

49. Potassium Tellurite media LJ media

50. Indicator media
These media contain an indicator which
changes its colour when a bacterium grows in
them.
Eg:
Blood agar
Mac Conkey’s medium
Christensen’s urease medium

52. Urease medium

53. Differential media
A media which has substances incorporated in
it enabling it to distinguish between bacteria.
Eg: Mac Conkey’s medium
Distinguish between lactose fermenters & non
lactose fermenters.

54. Lactose fermenters – Pink colonies
Non lactose fermenters – colourless colonies

55. Transport media
Media used for transporting the
samples.
Delicate organisms may not
survive the time taken for
transporting the specimen without
a transport media.
Eg:
Stuart’s medium – non nutrient soft
agar gel containing a reducing agent
Buffered glycerol saline – enteric
bacilli

56. Anaerobic media
These media are used to grow anaerobic organisms.
Eg: Robertson’s cooked meat medium, Thioglycolate
medium.

58. Growth in Continuous Culture
• A “continuous culture” is an open system in which fresh
media is continuously added to the culture at a
constant rate, and old broth is removed at the same
rate.
• This method is accomplished in a device called a
chemostat.
• Typically, the concentration of cells will reach an
equilibrium level that remains constant as long as the
nutrient feed is maintained.

61. Serial dilution Method of
Bacterial Enumeration

62. Many studies require the quantitative
determination of bacterial populations. The two
most widely used methods for determining
bacterial numbers are:
I. The standard plate count method.
II. Spectrophotometer (turbid metric) analysis.
The standard plate count method is an indirect
measurement of cell density ( live bacteria).
The spectrophotometer analysis is based on
turbidity and indirectly measures all bacteria (cell
biomass), dead and alive.
Introduction

63. The Plate Count (Viable Count)
However, if the sample is
serially diluted and then
plated out on an agar surface
in such a manner that single
isolated bacteria form visible
isolated colonies, the number
of colonies can be used as a
measure of the number of
viable (living) cells in that
known dilution.
The number of bacteria in a given sample is usually too great
to be counted directly.

64.  Keep in mind that if the organism normally
forms multiple cell arrangements, such as
chains, the colony-forming unit may consist of a
chain of bacteria rather than a single bacterium.
In addition, some of the bacteria may be clumped together.
Therefore, when doing the plate count technique, we generally
say we are determining the number of Colony-Forming Units
(CFUs) in that known dilution.
By extrapolation, this number can in turn be used to calculate
the number of CFUs in the original sample.
bacterial counts by these methods are usually expressed as
colony forming units per milliliter (CFU/mL).

65. Normally, the bacterial sample is diluted by
factors of 10 and plated on agar.
After incubation, the number of colonies on a
dilution plate showing between 30 and 300
colonies is determined.
A plate having 30-300 colonies is chosen because this range is
considered statistically significant.

66.  If there are less than 30 colonies on the plate, small
errors in dilution technique or the presence of a few
contaminants will have a drastic effect on the final
count. (too few to count (TFTC).
 Likewise, if there are more than 300 colonies on the plate, there
will be poor isolation and colonies will have grown together. (too
numerous to count (TNTC).

67. Procedure
1) Using sterile technique, transfer 1 mL
sample to the first dilution blank. Mix the
bottle by inverting it 20 times. Label the
bottle "10-1."

68. 2) Using a fresh pipette, transfer 1 mL
from the first blank to the second
blank. Mix as before. Label the second
bottle "10-2."3) Using a fresh pipette, transfer 1 mL from the first blank to the
second blank. Mix as before. Label the second bottle "10-2“.
4) Using a fresh pipette, transfer 1 mL from the first blank to
the third blank. Mix as before. Label the second bottle "10-3“.
5) Using a fresh pipette, transfer 1 mL from the first blank to the
forth blank. Mix as before. Label the second bottle "10-4“.
6) Using a fresh pipette, transfer 1 mL from the first blank to the
second blank. Mix as before. Label the fifth bottle "10-5“.
7) Label the Petri dishes: 10-2, 10-3, 10-4, 10-5, and 10-6,
respectively.

69. 8) transfer liquid from the dilution
blanks to the Petri dishes. Use a
separate pipette for each blank, not for
each plate (i.e. if more than one plate
uses liquid from a single blank, a
single pipette may be used for that
blank).
9) One at a time, add a tube of molten nutrient agar to each
Petri dish. After adding the agar, gently swirl the dishes in
pattern for 30 seconds to mix the bacteria with the agar.
10)After the agar has thoroughly solidified, incubate the plates
at 37°C for 24 to 48 hours.
11) Count the number of colonies on a plate that has between 30
and 200 colonies. Any plate which has more than 200
colonies is designated as "too numerous to count" (TNTC).
Plates with fewer than 30 colonies do not have enough
individuals to be statistically acceptable.

72. Colonies Forming Units {CFU}
Calculate the number of bacteria (CFU) per
milliliter or gram of sample by dividing the
number of colonies by the dilution factor
multiplied by the amount of specimen added
to agar plate.
To compute the number of CFU/mL, use the
formula:
 c = concentration, CFU/mL
 n = number of colonies

74. CFU Calculation Example
 You count 46 colonies on your plate
 You put 1 ml of bacterial culture into 99 ml of saline
and plated 0.1 ml
 Dilution 1/100
CFU= 46
1/100 * 0.1
= 46 * 100 * 10 =46000 CFU/ml