Fundamentals of Microbiology Guidance : Dr. Chandrika Nanayakkara ( Senior Lecturer, Dept. Head , Department of Plants Sciences, University of Colombo

1. BT 2014
Principals of Microbiology

2.  Cell Morphology
1. Size
2. Shape
i. coccus
ii. Rods
iii. Helical
iv. Spiral

3. • Consists with,
1. Cell membrane/ Plasma membrane
2. Cell wall
3. Chromosome
4. Plasmids
5. Ribosomes
6. Inclusion bodies – food granules
7. Bacterial chloroplasts
8. Flagella
9. Fimbriae
10. Pilli

4. • Total everything that inside the plasma membrane.
• Gel like
• Molecules moves rapidly through it – cytoplasmic streaming
Components in the cytoplasm
1. proteins – enzymes
2. ribosomes – RNA & protein
3. storage granules – depending on
its physiology
nutritional environment
4. waste materials – alcohol, lactic & acetic
acids
Functions: place for metabolism, cell growth and replication

5.  Large circular genome – DNA
 Instruction manual for built a particular bacteria
 Contains genetic info.
 Needed for survive and produce daughter cells.

6.  Small circular DNA
 Replicate independently of the chromosome
 Not essential for survival
 Selective advantage
 Carry extra genes
 1-100 in a cell

7.  Total structure that defines the exterior of the cytoplasm.
 Includes,
1. cytoplasmic membrane
2. 1 or 2 other layers
 Essential for viability
 Most important site that attack by the antibiotics
 Ligands – adherence and receptors – drugs and viruses
 Immunological distinction and variations - classification
Functions :
• protect from mechanical damage
• osmotic rupture
• lysis

8. •Bacteria are usually live in relatively dilute environment because the solute concentration inside
the cell is more high.
Osmotic pressure(inside) – (10 – 25) atm
Murein / Peptidoglycan – ubiquitos component of bacteria cell wall.
Cell wall chemistry
Staining characters Sensitivity to drugs

9. Gram’s classification
Gram positive
•Thick cell wall composed
of primarily peptidoglycan
Gram negative
•Two layers, thin
peptidoglycan layer and
outer membrane

10. Gram’s reaction
Gram positive
Retain initial dye –
crystal violet
Gram negative
Decolorize
Counterstain by
safranin
purple pink
Gram variable
Varying
result

11.  Bacterial murein – a unique type of peptidoglycan
 Peptidoglycan – polymer of sugars (glycan) cross linked by short chains of amino
acid(peptide)
 peptidoglycan contains,
1. N-acetylmuramic acid (NAM)
2. N-acetylglucosamine (NAG)
Alternatively.
Long strands of the alternating polymer linked
by,
1. L – alanine
2. L – lysine
3. D- alanine
4. D – glutamic acid
Tetrapeptides to
NAM
Peptidoglycan is a target for antibacterial activities.
 penicillin – synthesis enzymes interrupt.
Lysozyme – NAM – NAG bond interrupt.

12. highly a crossed peptidoglycan structures than gram negative.
40 – 100 s of peptidoglycan sheets in the layer.

13.  Teichoic acids are covalently connected with N-acetylmuramic acids and make an
interconnection between linked peptidoglycan sheets.
 Consist with glycerol phosphate or ribitol phosphate.
 Only found in gram positive bacteria cell wall.
 Polyalcohol are connected by phosphate easter and typically have D – alanine and sugars.
 Lipoteichoic acids (LTA) – teichoic acids that extends through the entire polysaccharide layer
and bonded to the lipids in the plasma membrane. Antigenic determinant.

14. Functions :
1. Make inter connections between peptidoglycan sheets.
2. Bind Ca2+ and Mg2+ for eventual transport into the cell.

15.  Consist with two main layers
1. Inner thin peptidoglycan layer
2. Outer membrane

16.  Outer membrane
The outer most layer of the Gram negative bacteria cell wall.
Attach to the thin peptidoglycan layer by continuous layer of lipoprotein molecules by using
covalent bonds.
Function : protective barrier, prevent and slow the entry of bile salts, antibiotics & toxic, prevent loss of constituents
like periplasmic enzymes.

17.  Lipopolysaccharides(LPS)
• found in the upper part of the Gram negative bacteria cell wall outer membrane.
• composed of 3 parts.
1. Lipid – A
2. Core polysaccharide
3. O Polysaccharides (O antigen)
Lipid – A : binds with the outer membrane. Endotoxin portion of the molecules.
Core polysaccharide : consists of ketodeoxyoctonate (KDO)various heptoses and N-
acetylglucosamine.
O antigen – polysaccharides appear on the cell surface. Antigenic determinant.

18.  Porin proteins
 Small molecules such as molecules with 600 to 700 daltons in size can move through the outer
membrane via porin proteins.
 Porin proteins is a cluster of 3 porin molecules which make a narrow passage between outside
and the periplasmic space.
 Involved in trans membrane transportation of small hydrophilic molecules.

19. ֎ Periplasmic space
• the space between the outer membrane and the inner
membrane.
• contains with,
1. loose network of peptidoglycan chains
2. gel containing hydrolytic enzymes and
digestive enzymes.
3. other enzymes (peptidoglycan synthesis,
electron transport, toxic substance alternation)
• some species contained β- lactamalase enzyme in here.
Beta-lactamases (β-lactamases) are enzymes produced by bacteria that
provide multi-resistance to β-lactam antibiotics such
as penicillins, cephamycins, and carbapenems.

21. • included in the acid fast bacteria who are rich in mycolic acid.
• a thin layer of peptidoglycan covers the cell membrane.
• The peptidoglycan is attached to other sugars, galactan and arabinan. These sugars connect
the peptidoglycan to the identifying characteristic of all acid fast bacteria - mycolic acids.
• consist with approximately 60% of lipids.
• slow growth due to the impermeable effect of lipids in the acid fast envelop to the nutrients.

22. ֎ Acid fast stain - Process
Mycoplasma ,Thermoplasma are the archea without cell wall. They have tough outer
membranes and protected habitats from osmosis.

23. •A gelatinous structure that covers the entire bacterium which is made by polysaccharides.
•Identified by negative staining techniques.
1. Firmly attached – capsule
2. Loosely attached – slime layer
Functions :
1. Attachment
2. Protection from phagocytosis
3. Resist desiccation

24. •The most common type of cell wall in archea and many bacteria.
•Consists of interlocking monomolecules of proteins or glycoproteins.
•Self assemble two dimensional array of proteins (10-15% of total proteins in the cell)
•Thickness depends on the species.

25. Functions of S - Layer
•Protective outer layer for most archea and many bacteria.
•Protection against bacteriophage and phagocytosis
•Low pH resistency
•Lytic enzymes to prevent from heavy molecules
•Adhension
•Cell membrane stabilization
•Adhension sites for exo proteins
•Provides periplasmic components

26. Growth – increase of cell number
Colony – aggregation of cells arose from division of a single cell.
•Binaryfission
The method of bacterial cell growing
Bacterial cell
Grow in size Division (duplication)
Population density: growth in terms of cell numbers

27. Time taken for a bacteria cell to grow, divide and form two daughter cells.
The growth cycle depends on,
1. environmental factors
2. nutrition
3. genetic factors

28. • most chemical and physical factors are affected. mainly four,
1. Temperature
2. pH
3. Water availability / osmotic potential
4. Air / Oxygen

29. Cardinal temperatures
• minimum – below this level no longer occurring growth
• optimum – high growth rate
• maximum – above this level cells no longer exist
•Cardinal temperature ranges are varying according to the species.
eg: Pyrolobus fumari – maximum 113° C

30. There are four temp. classes in the bacteria
1. Psycrophiles
2. Mesophiles
3. Thermophiles
4. Hyperthermophiles
Biotechnological uses of thermophiles:
 enzymes functioning at high temp. used in industries.
eg: Thermus aquaticus – Taq polymerase (heat stable enzyme)

31. Mostly the optimum pH value is distributed around the pH 7.
But in some cases,
extreme acidophiles
extreme alkaliphiles
Bacteria secrete some extra cellular enzymes with optimum p H ranges.

32. Water availability depends on,
water content
concentration of dissolved solutes

33. No facultative aerobes

34. •Fts proteins – Filamentous temperature sensitive proteins
•Universally distributed
•Found in mitochondria and chloroplasts
•Structural similarities to the tubulin.

35. • Fts proteins in bacteria cell division
2 chromose copies pulled apart Constriction occurs
Fts-Z depolymerizeTriggers inward growth of wall
material
2 new cells

36. Growth rate : change in cell no. or mass per unit time
Generation time : time taken to double the population size
Generation time = Doubling time
Calculation of generation time
Cell number
1  2 21
2  4 22
3  8 23
n  x 2n
N – final cell number
N0 – initial cell number
n – no. of generations

38. Has four phases.
• Lag phase
• Log phase
• Stationary phase
• Death phase

39. Counts : Direct, Indirect, Total and Viable counts
1) Direct Counts
 Microscopic counts
Total count is obtained.
Dried Samples – Glass slide
Samples in solutions – Counting chambers
eg:- 0.1 µl / sq.
Using microscpic slide,
1. Mark 1 x 1 cm2 area.
2. Spread the known volume over the
area.
3. determine the vol. of a
microscopic area under high power.
4. cont the no. of microbes.
5. get the mean
6. convert to cells/ml
d
h

40.  Disadvantages
1. Viable (able to divide) or not
2. small cells
3. lack contrast (stain) , phase contrast
4. low density
5. motile microbes – 4% formaldehyde can fix
6. counting edge overlaps

41.  viable count
Viable cells - cells with ability to divide
No. of cell colonies (colony count / plate count)
Assumption – each viable cell yield one colony.
Two methods,
i. Spread plate
ii. Pour plate
Dilution for viable count
methods

42. Advantages Disadvantages
Easy, simple and viable counts have taken Dilute samples
Use of selective media No. of colonies should be 30 – 300
Suitability of medium / pH / temp.
Length of incubation
2 or more cells in a clump. (cfu / ml)
2) Indirect measurements
Turbidity
• an estimation of cell no.
• cell suspension scatter light
• more cells – more scatterings – turbid
• turbidity can be measured by using turbid meter, photometer or
spectrometer.
• it detect the amount of light scattered.

43. Optical density (OD) Vs. Cell no.
For unicellular microbes,
OD α Cell no.
•Turbidity can be considered as an indirect measurement of cell no.
Transmittance and Absorbance
Sample
P0 P