Identification technique Bacterial Growth curve Bacterial reproduction

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

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

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

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

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

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

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 +

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

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

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

Observed Under 10X Gram +Ve Gram -Ve

Observed Under 45X Gram +Ve Gram -Ve

Observed under 100X Gram +Ve Gram -Ve

Mechanism of Action of Gram staining

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

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

After adding Saffranin Small size pores Big size pores After adding Saffranin

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

Observed under 100X Gram +Ve Gram -Ve

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

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

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

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

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

Growth curve

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

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

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

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.

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

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

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

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

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.

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

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)