Bacteria – Morphology & Classification

1. Bacteria – Morphology & Classification II MBBS Dr SathyaAnandam Assistant prof, Microbiology

2. Introduction: • Based on the organization of their cellular structures, all living cells can be divided into two groups: eukaryotic and prokaryotic • Eukaryoticcell types - Animals, plants, fungi, protozoans, and algae • Prokaryotic cell types - bacteria & blue green algae

3. Schematic of typical animal (eukaryotic) cell, showing subcellular components. Organelles: (1) nucleolus (2) nucleus (3) ribosome (4) vesicle (5) rough endoplasmic reticulum (ER) (6) Golgi apparatus (7) Cytoskeleton (8) smooth ER (9) mitochondria (10) vacuole (11) cytoplasm (12) lysosome (13) centrioles

4. Prokaryotic Cells • much smaller (microns) and more simple than eukaryotes • prokaryotes are molecules surrounded by a membrane and cell wall. • they lack a true nucleus and don’t have membranebound organelles like mitochondria, etc. • large surface-to-volume ratio : nutrients can easily and rapidly reach any part of the cells interior

5. Differences between prokaryotic & eukaryotic cells

6. Differences between prokaryotic & eukaryotic cells

7. Differences between prokaryotic & eukaryotic cells

8. Size of Bacteria • Unit of measurement in bacteriology is the micron (micrometre, µm) • Bacteria of medical importance • 0.2 – 1.5 µm in diameter • 3 – 5 µm in length

9. Shape of Bacteria • Cocci – spherical/ oval shaped major groups • Bacilli – rod shaped • Vibrios – comma shaped • Spirilla – rigid spiral forms • Spirochetes – flexible spiral forms • Actinomycetes – branching filamentous bacteria • Mycoplasmas – lack cell wall

10. Other shapes of bacteria Comma shaped Spirilla Spirochetes

11. Arrangement of bacteria: Cocci Coccus Cocci in pair – Diplococcus Tetrad – groups of four Cocci in chain - Streptococci Sarcina – groups of eight Cocci in cluster - Staphylococci

12. Arrangement of bacteria: Bacilli

13. Anatomy of a Bacterial Cell

14. Anatomy of A Bacterial Cell • Outer layer – two components: • Rigid cell wall • Cytoplasmic (Cell/ Plasma) membrane – present beneath cell wall • Cytoplasm – cytoplasmic inclusions, ribosomes, mesosomes and nucleus • Additional structures – plasmid, slime layer, capsule, flagella, fimbriae (pili), spores

15. Structure & Function of Cell Components

16. CELL WALL • Outermost layer, encloses cytoplasm • Confers shape and rigidity • 10 - 25 nm thick • Composed of complex polysaccharides (peptidoglycan/ mucopeptide) - formed by N acetyl glucosamine (NAG) & N acetyl muramic acid (NAM) alternating in chains, held by peptide chains.

17. Cell Wall • Cell wall – • Carries bacterial antigens – important in virulence & immunity • Chemical nature of the cell wall helps to divide bacteria into two broad groups – Gram positive & Gram negative • Gram positive bacteria have simpler chemical nature than Gram negative bacteria. • Several antibiotics may interfere with cell wall synthesis e.g. Penicillin, Cephalosporins

18. Gram positive cell wall The Gram-positive cell wall is composed of a thick, multilayered peptidoglycan sheath outside of the cytoplasmic membrane. Teichoic acids are linked to and embedded in the peptidoglycan, and lipoteichoic acids extend into the cytoplasmic membrane

19. Gram negative cell wall The Gram-negative cell wall is composed of an outer membrane linked to thin, mainly single-layered peptidoglycan by lipoproteins. The peptidoglycan is located within the periplasmic space that is created between the outer and inner membranes. The outer membrane includes porins, which allow the passage of small hydrophilic molecules across the membrane, and lipopolysaccharide molecules that extend into extracellular space.

20. Cell Wall

21. Property of bacteria Gram Positive Gram Negative Thickness of wall 20-80 nm 10 nm Number of layers in wall 1 2 Peptidoglycan content >50% 10-20% Teichoic acid in wall + - Lipid & lipoprotein content 0-3% 58% Protein content 0% 9% Lipopolysaccharide 0 13% Sensitive to penicillin Yes Less sensitive Digested by lysozyme Yes Weakly Summary of the differences between Gram positive & Gram negative bacteria

22. Demonstration of cell wall. • Plasmolysis: In hypertonic environment the water diffuses from the bacterial cell and the cytoplasm shrivels up and pulls away from the cell wall. • Microdissection • Reaction with specific antibodies • Mechanical rupture of the cell: ultrasonic waves. • Differential staining procedures • Electron microscopy

23. CELL WALL –DEFICIENT ORGANISMS • They are bound by soft trilaminar unit membrane containing sterols. • Lack cell wall they are highly plastic and they can pass through bacterial filters. • Mycoplasma and Ureaplasma • They lack the cell wall precursors like muramic acids or diaminopimelic acid.

24. L-FORMS • Kleineberger-Nobel, named them L-forms after LISTER INSTITUTE London. • These are abnormal growth forms developed either spontaneously or in the presence of penicillin that interfere with cell wall synthesis. • They differ from parent bacteria in lacking rigid cell wall. • They are capable of growing and multiplying.

25. PROTOPLASTS AND SPHEROPLASTS: • When a cell is subjected to hydrolysis with lysozyme results in the removal of cell wall. • If the media is osmotically protective it results in the liberation of protoplasts from Gram positive cells. • Spheroplasts are produced by gram negative cells which retain their outer membrane.

26. Cytoplasmic (Plasma) membrane • Thin layer 5-10 nm, separates cell wall from cytoplasm • Acts as a semipermeablemembrane: controls the inflow and outflow of metabolites • Composed of lipoproteins with small amounts of carbohydrates

27. Functions: • Permeability and transport: active, passive and group translocation • Electron transport and oxidative phosphorylation : cell respiration • Excretion of hydrolytic exoenzymes & pathogenicity proteins : breaks polymers in to units.

28. Cytoplasmic Matrix • Colloidal system of a variety of organic and inorganic solutes between the plasma membrane and the nucleiod. • Viscous watery solution • Packed with ribosomes • Highly organised

29. Other Cytoplasmic Components • Ribosomes – protein synthesis • Mesosomes – • Multilaminated structures formed as invaginations of plasma membrane • Principal sites of respiratory enzymes • Coordinate nuclear & cytoplasmic division during binary fission • More prominent in Gram +ve bacteria • Intracytoplasmic inclusions – reserve of energy & phosphate for cell metabolism e.g. Metachromatic granules in diphtheria bacilli

30. Demonstration: • Special stains: • Volutin granules: Alberts, Neissers, Ponders, Puchs • Polysacharide granules: Iodine • Lipid inclusions: Sudan Black

31. Nucleus • No nucleolus • No nuclear membrane • Genome – • single, circular double stranded DNA. • Haploid • Divides by binary fission • Demonstration: • Feulgen and HCL Geimsa : light microscope • Electron microscopy

32. Additional Organelles • Plasmid – • Extranuclear genetic elements consisting of DNA • Transmitted to daughter cells during binary fission • May be transferred from one bacterium to another • Not essential for life of the cell • Confer certain properties e.g. drug resistance, toxicity

33. Additional Organelles • Capsule & Slime layer – • Viscous layer secreted around the cell wall. • Polysaccharide / polypeptide in nature • Capsule– sharply defined structure, antigenic in nature • Protects bacteria from lytic enzymes • Inhibits phagocytosis • Stained by negative staining using India Ink • Can be demonstrated by Quellung reaction (capsule swelling reaction) • Slime layer – loose undemarcated secretion

34. Functions of capsule: • Antiphagocytic • Adherence • Resistance to dessication. • Excludes viruses and hydrophobic molecules

35. Capsule Demonstration • Staining • Positive: crystal violet, Hiss, Muir s • Negative : India ink • Electron microscopy • Serology- capsular antigens • Quellung reaction

36. Additional Organelles- Flagella • Long (3 to 12 µm), filamentous surface appendages • Organs of locomotion • Chemically, composed of proteins called flagellins • The number and distribution of flagella on the bacterial surface are characteristic for a given species - hence are useful in identifying and classifying bacteria • Flagella may serve as antigenic determinants (e.g. the H antigens of Gram-negative enteric bacteria) • Presence shown by motility e.g. hanging drop preparation

37. Ultra structure of Flagella: Threadlike locomotor appendages

38. Types of flagellar arrangement Polar/ Monotrichous – single flagellum at one pole ex: Vibrio Lophotrichous – tuft of flagella at one pole ex: Pseudomonas Amphitrichous – flagella at both poles. Ex: Spirillum Peritrichous – flagella all over. ex : E coli, Salmonella Amphilophotrichous – tuft of flagella at both ends

39. Demonstration: • Staining : wet mount method, Kirkpatrick, Leifsons ,Loefflers etc. • Electron microscopy

40. Additional Organelles • Fimbriae/ Pili – • Thin, hairlike appendages on the surface of many Gram-negative bacteria • 10-20µ long, acts as organs of adhesion (attachment) - allowing bacteria to colonize environmental surfaces or cells and resist flushing • Made up of proteins called pilins. • Pili can be of two types – • Common pili – short & abundant • Sex pili - small number (one to six), very long pili, helps in conjugation (process of transfer of DNA)

41. Demonstration: Electron microscopy. • Function: Attachment to solid surfaces Antigenic Agglutination to RBCs

42. Additional Organelles • Spores – • Highly resistant resting stages formed during adverse environment (depletion of nutrients) • Formed inside the parent cell, hence called Endospores • Very resistant to heat, radiation and drying and can remain dormant for hundreds of years. • Formed by bacteria like Clostridia, bacillus

43. The cycle of spore formation and germination At the beginning of spore formation, a septum forms, separating the nascent spore from the rest of the cell and all of the genetic material of the cell is copied into the newly-forming cell. The spore contents are dehydrated and the protective outer coatings are laid down. Once the spore is matured it is released from the cell. On germination, the spore contents rehydrate and a new bacterium emerges and multiplies.

44. Shape & position of bacterial spore Oval central Non bulging Spherical central Oval sub terminal Oval sub terminal Bulging Oval terminal Spherical terminal Free spore

45. Uses: • Bacillus stearothermophilus -spores • Used for quality control of heat sterilization equipment • Bacillus anthracis - spores • Used in biological warfare

46. Demonstration: • Unstained in Grams stain • Modified ZeihlNeelson, Malachite green, FuschinNigrosin • Unstained wet preparation in phase contrast microscopy

48. Pleomorphism & Involution forms • Pleomorphism – great variation in shape & size of individual cells e.g. Proteus species • Involution forms – swollen & aberrant forms in ageing cultures, especially in the presence of high salt concentration e.g. plague bacillus • Cause – defective cell wall synthesis

49. Microbial Growth - refers to the increase in number of cells, not the size of the cells Bacteria divide by binary fission

50. The interval of time between two cell divisions or the time required by the bacterium to give rise to two daughter cells under optimum conditions, is known as the generation time or population doubling time.