SEDIAAN BAKTERI

1. SEDIAAN BAKTERI

2. Specimen Preparation for Optical Microscopes Wet mounts and hanging drop mounts – allow examination of characteristics of live cells: motility, shape, and arrangement Fixed mounts are made by drying and heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts. 2

3. Figure 3.8Preparing a hanging drop mount. Wet mounts (to observe living microbes) - Simple wet mounts : dry out quickly - hanging drop mounts : Using petroleum jelly to prevent drying of microbe samples

4. Types of dyes - Basic dyes : with positive charges, stains the surfaces of most microbes - Acidic dyes : with negative charges, stains negatively charged parts of cells, including proteins ex) stain animal tissues that microbes have invaded - Mordants : intensify staining by increasing a cell’s affinity for a dye

5. Staining procedures Simple stains – use basic dyes to make cells visible Differential stains – to distinguish between types of microoganisms 1. primary staining 2. Destaining 3. counterstaining ex) Gram stain (Gram positive and negative bacteria) Primary stain (Gentian violet)  Mordant (Iodine)  Decolorization (ethanol)  Counterstain (safranin) Acid-fast stain (acid-fast bacteria : mycobacteria, some actinomycetes) Primary stain (Carbolfuchsin)  Mordant (heating with steam)  Decolorization (HCl + ethanol)  Counterstain (Methylene blue)

6. The Gram Stain Gram's Crystal iodine violet Decolorise with acetone Gram-positives appear purple Counterstain with e.g. methyl red Gram-negatives appear pink

7. Figure 3.9aGram-staining procedure.Steps of the procedure. • Special stains – reveal specific parts of a microbes, such as the wall, the nucleoid, an endospore, • membranes, flagella, or the capsule. • ex) flagella stains •  renders flagella visible by adding a material, making them thicker (tannic acid) •  thickened flagella are stained with dye (rosaniline) • negative stains •  Identify the presence of capsules

8. PEMBIAKAN DAN PERTUMBUHAN BAKTERI

9. Microbial Growth • Increase in the number of cells • Increase in microbial mass “Because individual cells grow larger only to divide into new individuals, microbial growth is defined not in terms of cell size but as the increase in the number of cells, which occurs by cell division."

10. Binary Fission 1 to 2 to 4 to 8 to ? • Asexual • Cell splits and replicated DNA goes with each part • Prokaryotes, Bacteria • + Fast and easy • - Everybody has the same DNA

11. Note nascent septum forming Binary Fission

14. Cell growth > 2000 chemical reactions • Some involve energy transformation • Other involve biosynthesis of • Small molecules => polymers => macromolecules => Cell structures • The General process • Duplication of DNA • Elongation of cell • Septum formation • cell-partition, result of growth of plasma membrane & cell-wall in opposing direction • Separation of two daughter cell

15. Cell growth • In some sp, separation of two daughter cell is incomplete • Linear chains: linked bacilli or cocci • Tetrads: cuboidal groups of 4 cocci • Sarcinae: groups of 8 cocci in a cubical packet • Grapelike clusters: staphylococci

16. Diplococcus Streptococcus

17. Tetrad Sarcinae

18. Staphylococcus

19. Growth duration • time require for a complete growth cycle is variable, • dependent on number of factors: nutritional & genetic • E. coli in the best nutritional conditions the time • (generation time) is about 20 min

20. Several proteins implicated in the cell-division process • Fts proteins (filamentous temperature sensitive) • Essential for normal cell-division & chromosome replication process in prokaryotes • FtsZ: • a key protein in the group • have even been found in mitochondria & chloroplasts

21. cell division: • divisome: • division apparatus of Fts proteins including FtsZ • FtsZ ring formation follows DNA replication • FtsZ ring defines division plane

22. FtsZ ring and cell division Appearance & breakdown FtsZ ring during E. colicel-cycle Phase contrast Nucleoid stain cell stained w/ specific FtsZ reagent Combination nucleoid + FtsZ staining • FtsZ proteins interact to form => Divisome • ring around middle cell (in yellow) • DNA synthesis stop  Fts Z ring formation between 2 DNA molecules • FtsZ ring depolymerize => inward growth of new membrane & wall material in both directions until a cell becomes twice its original length • Constriction: occurs to form 2-daughter cells

23. Peptidoglycan Synthesis and Cell Division • New wall formed before cell-division • At Fts Z ring: • Small openings in cell wall are created by autolysin (enzyme present in Divisome) • New cell material is simultaneously added (by bactoprenol) across the opening • Coordination is important so the cell does not leak (lysis)

24. Peptidoglycan Synthesis • Bactoprenol • Lipid carrier molecule • transports peptidoglycan building blocks across the membrane by rending precursor sufficiently hydrophobic • bonds to N-acetyl (glucosamine / muramic acid) / pentapetide peptidoglycan precursor • once in the periplasm: bactoprenol interacts w/ enzymes that insert cell-wall precursor into the growing point of the cell-wall & catalyze glycosidic bond formation

25. Peptidoglycan Synthesis Transpeptidation: • Final step in cell wall synthesis • Formation of peptide cross-links between muramic acid residues in adjacent glycan chains • G- diaminopimelic acid & D-Ala • G+L-Lys & D-Ala (interbridge) • Penicillin-binding proteins: in periplasma of G- • When penicillin binds to these proteins, no wall synthesis  continuous action of autolysins weakens the cell wall  lysis

26. Phases of Growth • Lag • Adapt to nutrients • Log • Active growth • Stationary • Death = Growth rate • Death • Nutrients consumed • pH too low (why?) • Optimize curves in production

28. Log Growth

29. Rapid Growth of Bacterial Population

31. Population Growth • Growth: • Increase in number of cells • Increase in microbial mass • Growth rate (μ): • Change in the number of cells/ unit of time • Generation (n): • Interval between two divisions • Generation time (g): • Time for population to double • during the exponential phase • Time between two cell- divisions Data for a population that doubles every 30 min. Data plotted on an arithmetic and a logarithmic scale The rate of growth of a microbial culture

32. Generation Time N = (log10Nf – log10No ) / .301 N Number of generations Nf Final Concentration of Cells No Original concentration of cells .301 Conversion Factor to Convert Log2 to Log10

33. What is a Logarithm? Logarithm is a functionthat gives the exponent in the equation bn = x. It is usually written as logbx = n. For example: 34 = 81 Therefore log3 81 = 4

34. Example 1N = (log10Nf – log10No ) / .301 Measure Culture at 9:00 a.m. 10,000 cells / ml Measure Culture at 3:00 p.m. 100,000 cells / ml Calculate N N = (log10Nf – log10No ) / .301 N = (5-4)/0.301 N = 1/0.301 N = 3.33 Generations in 6 Hours We Know: 6 Hours = 360 Minutes Therefore: Generation Time = 360 Minutes / 3.33 Generations N = 108 Minutes to Generate

35. Example 2N = (log10Nf – log10No ) / .301 Measure Culture at 9:00 a.m. 10,000 cells / ml Measure Culture at Noon 1,000,000 cells / ml Calculate N N = (log10Nf – log10No ) / .301 N = (6-4)/0.301 N = 2/0.301 N = 6.64 Generations in 3 Hours We Know: 3 Hours = 180 Minutes Therefore: Generation Time = 180 Minutes / 6.64 Generations N = 27 Minutes to Generate

36. Example 3N = (log10Nf – log10No ) / .301 Measure Culture at 9:00 a.m. 2000 cells / ml Measure Culture at 1:00 p.m. 18,000 cells / ml Calculate N N = (log10Nf – log10No ) / .301 N = (4.25-3.30)/0.301 N = .95/0.301 N = 3.16 Generations in 4 Hours We Know: 4 Hours = 240 Minutes Therefore: Generation Time = 240 Minutes / 3.16 Generations N = 75.9 Minutes to Generate N = 1.27 Hours to Generate

37. Exponential Growth • substrate and nutrients are abundant • growth rate : proportional to the number of microorganisms X = concentration of microorganisms at time t t = time  = proportionality constant or specific growth rate, [time-1] dX/dt = microbial growth rate, [mass/volume time]

38. Population Growth No = 5 x 107, Nt = 1 x 108, t = 2h → n = 1 generation → g = 2/1 → g = 2 h • Exponential Growth: • Population doubles per unit of time • # mo increases logarithmically: 1 → 2 → 4 → 8 → 16 → 32…2n) mathematically exponential growth Nt = N0 2n => log Nt = log N0 + n log2 => n = 3.3 (log Nt - log N0 ) • Nt = # cells at time t • N0 = # initial cells • n = # of generations during time (t) • Generation time (g): • directly from graph • derived from n  g = t / n • from the slope = 0.301/g • Growth rate constant (k): • measure # generation /unit time • k = ln 2 / g = 0.693 / g 5 x 107 g →g = 0.301/0.15 (slope) → g = 2 h Method of estimating the generation times (g) of exponentially growing populations with generation times of (a) 6 h and (b) 2 h from data plotted on semi logarithmic graphs.

39. Substrate Limited Growth m = maximum specific growth rate [day-1] S = concentration of limiting substrate [mg/L] Ks = Monod, or half-velocity constant [mg/L]

40. Substrate Utilization Y = substrate yield, [mass of biomass/mass of substrate consumed]

41. Bacterial Growth Can Be Modeled With Four Different Phases

42. Remember the Four Main Stages Lag Phase Initial Phase / Metabolic Activities Exponential Phase 2nd Phase / Optimum Growth / Doubling Stationary Phase 3rd Phase / Exhaustion of Nutrients / Accumulation of Wastes Death Phase Final Phase / Continued Accumulation 90% of Cells Die, then 90% of Remaining Cells Die, etc.

43. Phases of Microbial Growth

44. The Growth Cycle Lag-phase: mo adjusts new environment, synthesizes enzymes & essential constituents, repairs any lesions from earlier injury e.g. freezing, drying, heating. No cell-division occur. Exponential (Log) Growth Phase:Nt = N02n generation time (or time to doubling cell-number) is constant

45. The Growth Cycle Stationary Phase: Essential nutrient used up & waste & inhibitory products accumulate Many cell-functions may continue Reproduction (cell-division) & cell-death are balanced (No net increase cell-number) Death (decline) Phase: When the death rate exceeds the rate of reproduction Sometimes accompanied by cell-lysis Exponential decline phase

46. Quantification of Bacteria Cell Numbers Total Mass of the Population Population Per Media cells / ml or cells / gram Direct County Methods and Indirect Counting Methods

47. Measuring Growth • Direct Counts • Petroff-Hauser Chamber • Pro’s vs. Con’s • Serial Dilution • 10-fold serial dilutions • Pro’s vs. Con’s • MPN (Most Probable Number) • Put 10, 1, and 0.1 ml into 10-mls broth • Repeat 5 times per volume • Statistical accurate sampling • Public Health Standards are written for MPN

48. Direct Measurements of Microbial Growth: Total Counts • Estimation of total cell mass/number is essential in most studies involving growth (measure growth rate, • substrate utilization, effects of inhibitors as antibiotic) • Methods to determine cell mass • Direct (wet & dry weight) • Indirect • by chemical analysis of specific cellular component (nitrogen) • Total number of mo determine • Direct (direct counting or viability counting) • Indirect (turbidity)

49. Direct Counting Methods Normally Viable Counts Remember that a Colony Starts Out as 1 Bacteria that Reproduces Colonies May Not All Be The Same Size

50. Direct Measurements of Microbial Growth: Total Countsdirect microscopic counting using Petroff-Hausser counting chamber quick way of estimating cell number known volume of sample dried on slide & in counting chamber • Living & dead cells counted; Small cells difficult to see • Precision difficult to achieve • Phase contrast microscope required w/ no staining sample • Not suitable for cell-suspension at low cell density (sample concentration) • Motile cells has to be immobilized Limitation: