Bacterial physiology and genetics were discussed. Key points include: 1) Bacteria grow through an orderly increase in constituents and multiply through cell division. Growth is measured by a growth curve with four phases: lag, log, stationary, and decline. 2) Bacteria require nutrients, temperature, oxygen levels, and other environmental factors for growth. 3) Genetic variations occur through mutations, conjugation, transduction, and transformation. Plasmids and transposons allow horizontal gene transfer. 4) Staining techniques like Gram staining and acid-fast staining are used to differentiate bacteria based on cell wall structure.

1. BACTERIAL PHYSIOLOGY AND BACTERIAL
GENETICS
DR.NASRIN NAHAR
MBBS, BCS,MD(MICROBIOLOGY)
LECTURER
SHER-E-BANGLA MEDICAL COLLEGE
BARISHAL.

3. BACTERIAL GROWTH AND MULTIPLICATION
• Growth of Bacteria is the orderly increase of all the chemical constituents
of the bacteria.
• Multiplication is the consequence of growth.
• Death of bacteria is the irreversible loss of ability to reproduce.

5. GROWTH CURVE
• The growth curve indicates multiplication and death of bacteria.
• When a bacterium is inoculated in a medium, it passes through four
growth phases which will be evident in a growth curve drawn by
plotting the logarithm of the number of bacteria against time.
• These phases are:
• 1. Lag phase
• 2. Log phase or Exponential phase
• 3. Stationary phase
• 4.Decline phase or death phase.

6. GROWTH CURVE

7. Lag Phase:
• After a liquid culture broth is inoculated, the multiplication of
bacteria does not start immediately. It takes some time to
multiply.
• The time between inoculation and beginning of multiplication is
known as lag phase.
• In this phase, the inoculated bacteria become acclimatized to the
environment, switch on various enzymes, and adjust to the
environmental temperature and atmospheric conditions.
• During this phase, there is an increase in size of bacteria but no
appreciable increase in number of bacterial cells. The cells are
active metabolically.
• The duration of the lag phase varies with the bacterial species,
nature of culture medium, incubation temperature, etc.

8. IMPORTANCE OF LAG PHASE
• Protein synthesis inhibitor is effective in this phase.
• Membrane acting antibiotic can be used in this phase.
• Detergents, soaps and other surface acting agents act better in lag
phase.

9. Log phase
• This phase is characterized by rapid exponential cell growth (i.e.,
1 to 2 to 4 to 8 and so on).
• The bacterial population doubles during every generation. They
multiply at their maximum rate.
• This continues until one of the two things happens either one or
more nutrients in the medium become exhausted or toxic
metabolic products accumulate and inhibit growth.
• Growth rate is the greatest during the log phase.
• The log phase is always brief, unless the rapidly dividing culture
is maintained by constant addition of nutrients and frequent
removal of waste products.
• When plotted on logarithmic graph paper, the log phase appears
as a steeply sloped straight line.

10. IMPORTANCE OF LOG PHASE
• Cell wall inhibiting antibiotics acts during this phase.

11. Stationary phase
• After log phase, the bacterial growth almost
stops completely due to lack of essential
nutrients, lack of water, oxygen, change in pH of
the medium, etc. and accumulation of their own
toxic metabolic wastes.
• The count remains stationary due to balance
between multiplication and death rate.

12. IMPORTANCE
• Endospores start forming during this stage.
• Bacteria become Gram variable and show irregular staining.
• Many bacteria start producing exotoxins.
• Metachromatic granules formation occurs.
• Cell wall acting antibiotics may be used.

13. Decline phase
• During this phase, the bacterial population declines due
to death of cells.
• The decline phase starts due to
• (a) accumulation of toxic products and autolytic enzymes
and
• (b) exhaustion of nutrients.
• Involution forms are common in this stage. Some cells
assume various shapes, becoming long, filamentous rods
or branching or globular forms that are difficult to
identify.
• Some develop without a cell wall and are referred to as
protoplasts, spheroplasts, or L-phase variants (L-forms).

14. IMPORTANCE
• Exotoxin of C. dipththeria is produced in this phase.
• Endotoxin producing bacteria produce endotoxin in this stage.

15. BACTERIAL GROWTH REQUIREMENT
• Nutritional requirements:
• 1. Essential elements: Hydrogen and oxygen, Carbon and nitrogen,
Sulphur and phosphorus etc.
• 2.Growth factor:
• Essential: Vitamins: Thiamine, riboflavin nicotinic acid, pyridoxine,
folic acid and vitamin B-12.
• Accessory: Factor-X and Factor-V for H. influenza.
• 3. Mineral sources: Potassium, calcium, magnesium iron, copper,
cobalt, manganese, molybdenum and zinc.

19. CO2 requirements
• Small amounts of CO2 is required by all bacteria.
• Certain organisms grow best at a higher CO2 tension and are called
capnophiles, e.g., meningococcus, gonococcus etc.

20. TEMPARATURE

24. FERMENTATION OF SUGARS
• Anaerobic bacteria use compounds like nitrates or sulphates instead
of oxygen as final electron acceptors in the process of respiration
(anaerobic respiration).
• A more common process used by these bacteria in anaerobic
metabolism is fermentation.
• It is defined as the process by which complex organic compounds,
such as glucose, are broken down by the action of enzymes into
simpler compounds without the use of oxygen.
• This process leads to the formation of several organic end products
such as organic acids and alcohols, as well as of gas (carbon dioxide
and hydrogen).

25. NEXT TOPIC…….
BACTERIAL GENETICS

26. • Genetics: Genetics is the science that deals with the study of genes,
heredity, and genetic variation in living organisms. Thus, it is the study of
mechanisms by which different characteristics that are passed on from
parents to offsprings(progeny).
• Genome: Genome is the entire set of genes and thus is the totally of genetic
information in an organism.
• Gene: A gene is a sequence of DNA that codes for a known cellular function
or process.

32. MECHANISMS OF GENETIC VARIATIONS
• Mutations
• Transfer of genetic materials through:
• Conjugation
• Transduction
• Transformation
• Transposition

34. PLASMID
• A plasmid is a small, circular piece of DNA that is different than the
chromosomal DNA, which is all the genetic material found in an
organism’s chromosomes. It replicates independently of
chromosomal DNA.

35. Classifications and types
•Plasmids may be classified in a number of ways.
•Plasmids can be broadly classified into conjugative plasmids and non-
conjugative plasmids.
•Conjugative plasmids contain a set of transfer genes which promote sexual
conjugation between different cells.
•In the complex process of conjugation, plasmids may be transferred from
one bacterium to another via sex pili encoded by some of the transfer genes.
•Non-conjugative plasmids are incapable of initiating conjugation, hence
they can be transferred only with the assistance of conjugative plasmids.

36. • Another way to classify plasmids is by function. There are five main
classes:
Fertility F-plasmids, which contain tra genes. They are capable of
conjugation and result in the expression of sex pili.
Resistance (R) plasmids, which contain genes that provide
resistance against antibiotics or poisons. Historically known as R-
factors, before the nature of plasmids was understood.
Col plasmids, which contain genes that code for bacteriocins,
proteins that can kill other bacteria.
Degradative plasmids, which enable the digestion of unusual
substances.
Virulence plasmids, which turn the bacterium into a pathogen.

37. FUNCTIONS OF PLASMID
• Plasmid carry the genes for the following functions and structures of
medical importance:
• Antibiotic resistance, which is mediated by variety of enzymes.
• Exotoxins such as enterotoxin of E.coli, anthrax toxin of Bacillus anthracis,
exfoliative toxin of S. aureus and tetanus toxin of Clostridium tetani.
• Pilli which mediate the adherence of bacteria to epithelial cell.
• Resistance to heavy metals such as mercury, active component of some
antiseptics and silver, which is mediated by a reductase enzyme.
• Resistance to ultraviolet light, which is mediated by DNA repair enzymes
• Production of Bacteriocins, a toxic proteinproduced by certain bacteria that
are lethal for other bacteria.

40. RESISTANCE PLASMID

41. RESISTANCE PLASMID CONJUGATION

43. BACTERIOPHASE

50. TRANSPOSON
• Transposons are pieces of DNA that moves readily from one site to
another either within or between the DNAs of bacteria, plasmids, and
bacteriophases.
• Because of their unusual ability to move, they are nicknamed
“jumping genes”.
• Transposon can code for drug resistance enzyme, toxins, or a variety
of metabolic enzymes and can cause mutations in the gene into which
they insert or alter the expression of nearby genes.

51. TRANSPOSON

52. TRANSPOSON

59. MUTATION FUNCTIONALLY AFFECTS:
• Not able to produce capsule
• Antigenic structure alteration
• Drug resistance
• Altered colony morphology
• Altered pigment production.

61. • In case of aerobes, atmospheric oxygen is the final electron acceptor
in the process of respiration (aerobic respiration). In this case, the
carbon and energy source may be completely oxidised to carbon
dioxide and water. Energy is provided by the production of energy-
rich phosphate bonds and the conversion of adenosine diphosphate
(ADP) to adenosine triphosphate (ATP). This process is called
oxidative phosphorylation.

64. • Gram Staining Technique
• 1. Crystal violet acts as the primary stain. Crystal violet may also be used as
• a simple stain because it dyes the cell wall of any bacteria.
• 2. Gram’s iodine acts as a mordant (Helps to fix the primary dye to the cell
• wall).
• 3. Decolorizer is used next to remove the primary stain (crystal violet) from
• Gram Negative bacteria (those with LPS imbedded in their cell walls).
• Decolorizer is composed of an organic solvent, such as, acetone or ethanol
• or a combination of both.)
• 4. Finally, a counter stain (Safranin), is applied to stain those cells (Gram
• Negative) that have lost the primary stain as a result of decolorization

65. • RESULT
• Gram positive: Dark purple
• Yeast cell: Dark purple
• Gram negative: Pale to dark red
• Epithelial cell: Pale red
• Nuclei of pus cells: Red

67. • Acid-fast Stain
• In the acid-fast staining procedure, the red dye
• carbolfuchsin is applied to a fixed smear, and the slide
• is gently heated for several minutes. (Heating
• enhances penetration and retention of the dye.)
• Then the slide is cooled and washed with water. The
• smear is next treated with acid-alcohol, a decolorizer,
• which removes the red stain from bacteria that are not
• acid-fast.
• The acid-fast microorganisms retain the red color
• because the carbolfuchsin is more soluble in the cell
• wall lipids than in the acid-alcohol

68. • Acid-fast Stain
• In non-acid-fast bacteria, whose cell walls lack the
• lipid components, the carbolfuchsin is rapidly
• removed during decolorization, leaving the cells
• colorless.
• The smear is then stained with a methylene blue
• counterstain.
• Non-acid-fast cells appear blue after application of
• the counterstain.

69. • Gram Reaction
• Gram-positive bacteria are those that are stained dark blue or violet by Gram
• staining. This is in contrast to Gram-negative bacteria, which cannot retain the
• crystal violet stain, instead taking up the counter stain (safranin or fuchsine) and
• appearing red or pink. Gram-positive organisms are able to retain the crystal
• violet stain because of the high amount of peptidoglycan in the cell wall. Grampositive
• cell walls typically lack the outer membrane found in Gram-negative
• bacteria.
• Gram-negative bacteria are those bacteria that do not retain crystal violet dye
• in the Gram staining protocol. In a Gram stain test, a counter stain (commonly
• safranin) is added after the crystal violet, coloring all Gram-negative bacteria
• with a red or pink color. The test itself is useful in classifying two distinct types
• of bacteria based on the structural differences of their cell walls. On the other
• hand, Gram-positive bacteria will retain the crystal violet dye when washed in
• a decolorizing solution.

70. • Stain
• A stain is a substance that adheres to a cell, giving the cell color. The
presence
• of color gives the cells significant contrast so they are much more
visible.
• Different stains have different affinities for different organisms, or
different parts
• of organisms. They are used to differentiate different types of
organisms or to
• view specific parts of organisms