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1. PMB 241.2 INTRODUCTION TO PHARMACEUTICAL MICROBIOLOGY Dr. Chidozie N.E. Ibezim

2. Course content: Historical development of microbiology, scope and its relevance to Pharmacy Micro-organism and medicine – The discovery of micro-organisms; the role of micro-organisms in disease The scope and relevance of pharmaceutical microbiology; the future of pharmaceutical microbiology. Study of Microbial Structures – Microscopy; specimen preparations & examination methods-direct examination, differential, acid-fast & fluorescent stains. Presence and effects of micro-organisms on the environment - ubiquity; characteristics . Aseptic techniques – introduction/principles.

3. LEARNING OBJECTIVES To understand the origin of microbes, diseases and the relationship between them. To understand microscopy and its application to Pharmaceutical Microbiology. To understand relevance of microbiology and microorganisms in Pharmacy. To understand basic and fundamental techniques in Pharmaceutical microbiology procedures. To describe smear / sample preparation and staining principles.

4. DEFINITION OF MICROBIOLOGY Microbiology is the scientific and elaborate study of Microorganism ( ie living organisms of microscopic size). Microorganisms are minute, unicellular organisms, non-visible to naked eyes. These include bacteria, fungi, algae, protozoa and virus ( border line of life); their typical form, structure, reproduction, Physiological properties, metabolism, classification, distribution in nature, relationship and effects on humans, animals and plants, reactions to chemical and physical agents.

5. Pharmaceutical Microbiology is the an applied microbiology which relates to the study of microorganisms in relation to the production of antibiotics, enzymes, vitamins, vaccines and other Pharmaceutical products; as well as study of Microorganisms that cause P harmaceutical contamination and spoilage. Other applied microbiology include: Medical Microbiology Veterinary Microbiology Public health Microbiology Environmental Microbiology Industrial Microbiology Food and Diary Microbiology Agriculture Microbiology Water and Aquatic Microbiology Aero- Microbiology Microbial Biotechnology

6. Why study microorganisms? Microorganisms are ubiquitous, distributed everywhere with unique and specific features that enable them to adapt and survive in these environments as air, river, ocean beds, plant roots, ice, GIT, hot spring, oil wells etc . In microbial genetics it enables us understand how microorganism acquire genes for resistance and virulence. They are vital to the ecosystem because they are decomposers and recyclers. Microorganisms are used in the production of pharmaceuticals and complex drug molecules eg insulin and vaccines.

7. In food microbiology where they are utilized in the production of beers, wine, bread, cheese, etc. The study of microorganisms helps us understand the pathogenicity of diseases. Study of microorganisms enables us understand how living cells function. Most biochemical pathways work with microorganisms. Their control is essential in food safety, since they could cause putrefaction and disease. They are important symbionts

8. TRANSFER OF Microorganism Animal sources from human to human or human to animal via direct contact, indirect or intermediary animal hosts. Air borne discharged through mouth, nose, into the air, where it in turn settles on food, dishes or clothing. Food borne:- Pathogenic organisms can be transferred through food handling by infected persons, faecal or insect contamination. Contact infections:- Direct transmission of Microorganism through contact from one host to other as seen in Sexually transmitted diseases. Formites :- through inanimate objects e.g books, cloths etc. Human contacts, soil borne, insects etc

9. History of Microbiology Robert Hooke (1665):- made light microscope and named the cell by looking at cork; He wrote book micrographia called pictures of small stuff under microscope. Anton Von Leeuvenhoek (1674) :- first to see Microorganisms in pond water and bacteria in teeth scrapings. He observed that there were forms of life not visible to the naked eye He explained that microorganisms are ubiquitous ie they are found everywhere . Then came the theory of spontaneous generation which believes that life could originate from non-living or decomposing matter ie a theory of something coming from nothing. Example the existence of maggot on a piece of rag used to wrap bread

10. Spontaneous generation t heory (A theory of life from a non-life) Supported by John Needham (1745)----- by boiling nutrient broth, left it uncovered, resulted to tons of microbes in the broth. Lazzaro Spellazani (1765) ------- identified requirement of oxygen for microorganism to survive through his observation of microbial absence in boiled and covered water. However, this theory was refuted by: Francesco Redi (1668) – saying that something is coming from somewhere/something by his meat in jar experiment. Maggot was unable to grow on meat in the jar if meat was covered with gauze . Rudolf Virchow (1858)--- he developed the concept of biogenesis which states that living cells come from other pre existing cells

11. Ferdinand J. Cohn (1828-1898) One of the founders of bacteriology Started early research on unicellular algae Also rejected the doctrine of spontaneous generation Was the first to attempt the arrangement of different varieties of bacteria into genera and species on a systematic basis. Discovered the formation and germination of spores (endospores) in certain bacteria e.g Bacillus subtillis and its resistance to high temperature. This was demonstrated with quick reappearance of bacteria in thoroughly boiled flasks of hay and turnip–cheese infusions

12. Louis Pasteur (1861) & His Contributions: Totally discredited spontaneous generation theory He showed that microorganisms are not evenly distributed in the atmosphere but their number varies from place to place. “Pasteurization”---- Pasteur’s investigation on wine and beer fermentation and the finding that wine held for a few minutes at 50-60 c will not undergo spoilage gave rise to as a process of “pasteurization” usually employed in the preservation of milk, juice, wine, etc. He also demonstrated that different microorganisms do different work and under different environmental conditions . Introduction of the terms aerobic, anaerobic and facultative anaerobic organisms. Pasteur opened the field of sterilization and autoclaving by demonstrating that fluid material can be heated at 120 C under pressure (autoclaving) and that ordinary water had some microorganism even after boiling. He introduced the practice of sterilizing glass wares by dry heat at 170 C .

13. He demonstrated that air contains microorganism where he avoided air borne microorganism into nutrient broth plugged by cotton but trapped these organism in the S-necked flask. No growth was observed in the medium, instead on the cotton plug. If the necks were broken, dust would settle and the organisms would grow.

14. Germ theory This is known as GOLDEN AGE OF MICROBIOLOGY ( 1857-1914). It came following Pasteur’s experiment. The germ theory of disease, also called the pathogenic theory of medicine, is a theory that proposes that microorganisms are the cause of many diseases via their growth and reproduction. Although highly controversial when first proposed, germ theory was validated in the late 19th century and is now a fundamental part of modern medicine and clinical microbiology, leading to such important innovations as antibiotics and hygienic practices. Germ theory was demonstrated by: Agostino Bassi (1835): demonstrated that silk worm disease can be transferred from one silk worm to another by fungus. Thus suggested a link between microorganism and disease. J oseph Lister (1860) Known as the Father of antiseptic surgery . Applied germ theory to medicine. Lister introduced heat sterilization of surgical instruments as well as use of aseptic techniques for control of microbes using physical and chemical agents.

15. Robert Koch (1876): F ounder of medical bacteriology Confirmed evidence of Germ theory of disease. He isolated anthrax bacteria and established the relationship between Bacillus anthracis and anthrax. H is criteria became known as Koch’s Postulates and are still used to establish the link between a particular microorganism and a particular disease. Afterwards Koch’s assistant Richard Petri designed a special plate to hold solid culture media (Petri Plate ). It is a – must –found in microbiology field and it is known as Petri dish.

16. Koch’s Postulates A particular microbe (organism) must be found in association with a given disease . Causative organism must be isolated and cultivated in pure culture in the laboratory . The pure culture shall be able to cause the disease after being duly inoculated into a susceptible animal . The causative organism should be recovered from the infected experimental animal in its pure culture.

17. Contributions of Robert Koch Discovered the methods of staining bacteria, photographing and preparation of permanent slides . Development of solid culture media and methods for studying bacteria in pure cultures. Demonstrated that anthrax is caused by Bacillus anthracis . Use of agar as support medium for solid culture. Isolated Mycobacterium tuberculosis and vibrio cholera. He substantiated the germ theory of disease by relating a specific organism to the specific disease. He termed colonies (millions of one kind of bacteria). He also identified pure culture(an accumulation of one type of microorganism formed by growth of colonies of the organisms) He demonstrated that bacteria were temperature sensitive from the inability of chicken to acquire anthrax at their body temperature of 42 c but at lower temperature of 37 c.  

18. CHEMOTHERAPEUTIC ERA----BIRTH OF CHEMOTHERAPEUTIC AGENTS : Paul Ehrlich (1854 – 1915) titled Father of chemotherapy , laid the foundation of the era of chemotherapy. C hemotherapy is defined as the use of chemicals that selectively inhibit or kill pathogens without causing damage to victim. He in collaboration with Sakahiro Hata , discovered Salvarsan ( a drug) for the treatment of syphilis . Gerhardt Domagk R ecorded the first success in treating streptococcal infections upon discovery of azo-dye ( sulphonamide ) derived from paramino benzene sulphonamide with activity against streptococcus. Sir Alexander Fleming Accidentally discovered a substance produced by Penicillium notatum (a fungus) called penicillin with ability to destroy several pathogenic bacteria. Penicillium notatum was later replaced by Penicillium chrysogenum for the commercial production of penicillin during Second World War . S.A Waksman discovered streptomycin produced by two strains of Actinomycetes streptomyces griseus in 1944, thus paving way for more antibiotic discoveries from m icroorganisms .

19. DISEASES CAUSATIVE AGENTS DISCOVERER Anthrax Bacillus anthracis Robert Koch Actinomycosis Actinomyces bovis Bollinger AIDS HIV virus Lac montagnier Dysentery Shigella dysenteriae Kiyoshi shiga Meningitis Neisseria Meningitidis W eichselbaum Gas gangrene Clostridium perfringes William Welch Syphilis Treponema pallidium Schaudin Pneumonia Streptococcus pneumoniae Fraenkel Tetanus Clostridium tetani Arthur Nicolaier Botulism Clostridium botulinium Van Ermengem Whooping cough Bordetella pertussis Bordet & Gengou Q – fever Coxiella burnetii Derric Hepatitis A Hepatitis A virus Purcell et al Gastritis Helicobacter pylori Warren & Marshall Leukemia Human T-cell virus Robert Gallo et al Diphtheria Corynebacterium diptheriae Theodor Klebs Cholera Vibrio cholera Robert Koch Tuberculosis Mycobacterium tuberculosis Robert Koch Malaria Plasmodium spp Laveran Typhoid fever Salmonella typhii Eberth Gonorrhea Neisseria gonorrhea Eberth Neisser Leprosy Mycobacterium leprae Hansen TABLE 1: DISCOVERING OF CAUSATIVE AGENTS OF MICROBIAL DISEASES

20. Some common diseases caused by microorganisms

21. ANTIBIOTICS Antibiotic (Anti – “against", biotics – “life”): refers to any natural or synthetic substance that destroys microorganisms or inhibits its growth. Antibiotics are employed in the control, management and treatment of human infectious diseases; - Curing and controlling of plant and animal diseases. E.g Penicillin use in management and control of pests. -In animal husbandry as feed additive to cause enhancement in the fattening of food animals. -In food handling and processing to minimize spoilage of fish, vegetables and poultry products. -In research during the study of biochemical cellular mechanisms.

22. ANTIBIOTICS SOURCE DISCOVERER Aureomycin Streptomyces aureofaciens Dugger Amphotericin B Streptomyces nodosus Gold et al Aplasmomycin Streptomyces griseus SS-20 Okami et al Bacitracin Bacillus subtilis Johnson et al Chloramphenicol Streptomyces venezuelae Burkholder Cephalosporins Cephalosporin acremonium Giuseppe Brotsu Cycloserine Streptomyces species Kuchl et al Penicillin (unstable) Penicillium notatum Sir Alexander Fleming Nystatin Streptomyces noursei Hazen & Brown Erythromycin Streptomyces erythraeus McGuire et al Griseofulvin Penicillium griseofulvum Griseofulvin Penicillin (stable) Penicillium notatum Chain et al Neomycin Streptomyces fradiae Naksman & Lechevalier Vancomycin Streptomyces orientalis McCormick et al Kanamycin Streptomyces kanamyceticus Umezawa et al Rifamycin Nocardia mediterranei Sensi et al Gentamycin Micromonospora purpurea Weinstein et al TABLE 2: DISCOVERY OF ANTIBIOTICS

23. Modern developments in Microbiology and Pharmaceutical microbiology. Virology: study of biology of viruses and viral diseases Recombinant DNA technology/ genetic engineering using microbial genetics and molecular biology. Discovery of the role of mRNA in protein synthesis . Pharmaceutical biotechnology . Use as cloning vehicles ;as E.coli and other microbes have been employed extensively in cloning specific segments of DNA, large scale production of vital chemicals Immunology – study of immunity. Use of vaccines and interferons are being investigated for cure and prevention of viral diseases. Use of immunology as a diagnostic tool

24. Applications of Microbiology/Microorganism Biotechnology – this is the application of biology to solve practical problems and produce useful products economically. Food production – microorganisms ferment grains to produce beverages, they also ferment milk (dairy) to produce cheese and yoghurt. Bioremediation – this is the use of bacteria to destroy chemical pollutants eg polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT), trichloroethylene. Bacteria are also used to clear up oil spills. Bacteria are used to synthesize products for industrial use – ethanol, chemicals poisonous to insects, cellulose, amino acids.

25. Genetic engineering – the introduction of genes from one organism to an unrelated organism to confer new properties. Microorganisms are genetically engineered to produce medically important products eg insulin, human growth hormones, enzymes. Microorganisms are modified to produce vaccines eg gonorrhoea, hepatitis etc. Viruses are studied to be used to deliver genes into humans to correct conditions like cancer, etc.

26. Tools for the exploration of basic life phenomenon. They serve as basic research- role - model to various life processes. This is because of the unique properties of these organisms namely:- -extreme fast reproduction. -ability to be cultured (grown) quickly or conveniently either in small or large quantum. - Their growth may be easily manipulated and monitored using chemical and physical methods. Their cells may be cleared and torn apart, and the contents segregated into different fractions of varying particle sizes.

27. Scope and Relevance of Pharmaceutical Microbiology Pharmaceutical microbiology finds its application in the study of microorganisms associated with the manufacture of pharmaceuticals. Minimizing the number of microorganisms present in a process environment. Excluding microorganisms and their by products like exotoxins and endotoxins from water and other starting materials Ensuring that a finished pharmaceutical product is free from contaminating organisms. Research and development. Development of anti-infective agents (drug discovery). Use of microorganisms to determine the mutagenic or carcinogenic activity of prospective drugs. Use of microorganisms in manufacture of pharmaceutical products eg insulin and human growth hormones

28. Scope and relevance cont’d It is important in antimicrobial activity and disinfection; to determine how a product will act in cases of contamination. This is achieved by challenging the finished product with potential contaminants as part of quality control tests. Understanding the mechanisms of resistance of pathogenic organisms to various antimicrobial agents and how they can be overcome.

29. STUDY OF MICROBIAL STRUCTURES MICROSCOPY TECHNIQUES USED IN MICROBIOLOGY Types of microscope: Light microscope Bright field microscopy Phase contrast microscopy Dark field microscopy Fluorescence microscopy ELECTRON MICROSCOPY Scanning electron microscopy (SEM) Transmission electron microscopy (TEM) Light microscope uses visible light for observing objects. It can magnify objects up to 1000 times making it easier to observe the shape, size and motility of cells.

30. Comparing light and electron microscopes

31. Definition of some terms Magnification This is the enlargement of the object being viewed. The microscope uses the ocular and the objective lens for magnification. Total magnification is the product of magnification of both lenses (ocular and objective). Ocular magnifies object 10x while objective magnifies 4x, 10x, 40x and 100x. 100x lens is called the oil immersion objective; it gives total magnification of x1000 . Oil immersion lens increases resolution. Resolution Resolution refers to the ability of a lens to separate or distinguish small objects that are close together . It enhances distinctiveness, makes the object being observed not to be blurred.

32. Resolving Power Resolving power is the minimum distance existing between two objects when the objects can be viewed as distinct entities. Higher resolving power translates to greater detail. Maximum resolving power of the best microscope is 0.2 µm . It depends on type of lens, wavelength of light, s pecimen preparation and level of magnification Contrast This enables an object to stand out in the background. High contrast means that there is a high level of distinctiveness between the different shades/colours in a specimen. Different levels of contrast is required for different specimens to reveal the most information. Contrast is achieved in bright field microscopy by the use of staining techniques.

33. BRIGHT FIELD MICROSCOPE

34. Bright field microscope is the most commonly used light microscope available. It produces a dark image against a brighter background. The Dark-field Microscope Produces a bright image of the object against a dark background. It is used to observe living, unstained preparations . Organisms viewed with this microscope stand out as a bright contrast against a dark background . Dark field microscopy is ideal for reflectively labelled specimens and in delineating plant cell walls on unlabelled tissue sections. It can be used in the detection of Treponema pallidum , the causative organism of syphilis.

35. Phase contrast microscope It amplifies the slight difference between the refractive index of cells and surrounding media resulting in a darker appearance of the denser material. When beams of light pass through a specimen, they are partially deflected based on the difference in their densities or thickness and their refractive index. Phase contrast Microscope

36. It gives a more detailed cellular structures than bright-field microscopy. Living cells are viewed in phase contrast without killing them as with staining in bright field. The principle to make phase changes visible in phase-contrast microscopy is the ability to separate the illuminating (background) light from the specimen-scattered light (which makes up the foreground details ).

37. Fluorescence microscopy Uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances.  It is used to observe organisms that fluoresce naturally or have been tagged with fluorescent dyes. It gives a high intensity of contrast. The fluorescence microscopes projects UV light through a specimen, but then captures only light emitted by the fluorescent molecule to form the image . Principle: Employs the property of some molecules that when they are hit by a photon, they can absorb the energy of that photon to get into an excited state. Upon relaxation from that excited state, the same molecule releases a photon: fluorescence emission. Molecules (Dyes), called fluorochromes / fluorophores get excited when they absorb ultraviolet rays causing its energy level to increase thereby emitting light of a longer wavelength which is visible. Useful to observe total cells, subset of cells and cells with certain proteins on their surface

38. Basic characteristics: 1. Use of a very powerful light source 2. Utilization of specialized filters, like the excitation filter and the emission filter. Fluorescence Microscope

39. Fluorescence photomicrograph Fluorescent microscopy can be divided into fluorochroming and immunofluorescence In Fluorochroming , dyes interact with components of the microbial cell to give a great contrast and therefore increases the ability of observer to detect cells. It is used to confirm the presence of bacteria in blood cultures when Gram stain is difficult to interpret or when the presence of bacteria is suspected but not detected with bright field microscopy. E.g in the identification of Trichomonas vaginalis. Acridine orange binds to nucleic acids and i t stains bright orange. In the identification of mycobacteria, Auramine and rhodamine having affinity for the mycolic acids in the cell wall of Mycobacteria , causes Mycobacteria to appear bright yellow or orange against a greenish background In Immunofluorescence, fluorescent dyes are linked with antibodies to form an antibody-dye conjugate. Here, there is a combination of high contrast provided by fluorescence with specificity of antibody-antigen binding. It can be used to examine patients’ specimens for difficult to grow organisms eg Chlamydia, Bordetella pertussis.

40. ELECTRON MICROSCOPY Electron microscope uses electromagnetive lens, beam of electrons and a fluorescent screen to produce the magnified image unlike light microscope that uses glass lens, light and the eye to observe specimens. Developed by Knoll and Ruska It magnifies an object up to 100000x. The resolving power increases to about 1000 fold since electrons have a wavelength of about 1000 times shorter than visible light , giving a much higher resolution than the light microscope . However, the lenses and specimens must be in vacuum thus making it expensive and bulky and it also requires complex specimen preparation. They are used to investigate the ultra structure of a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. In electron microscopy, the instrument directs a beam of electrons at a specimen. Depending on the density of a region of the specimen, the electrons will either pass through or be scattered to varying degrees.

41. Types: Scanning electron microscope (SEM ): SEM is used for observing surface details of cells. Here, the beam of electrons scans back and forth over the surface of the specimen. Transmission electron microscope (TEM): TEM is used to observe fine details of cell structure such as number of layers that envelope a cell. Reflection electron microscope ( REM ): REM is a combination of imaging, diffraction, and spectroscopy techniques for characterization of topography, crystal structure, and composition of surfaces of single crystals. REM is not commonly applied in the study of microbial structures.

42. SPECIMEN PREPARATION Microorganisms are normally stained with dyes to immobilise them and make them visible using a brightfield microscope. Different staining techniques are used with their specific applications Basic dyes are more commonly used for staining than acidic dyes because basic dyes are positively charged. Basic dyes are attracted to negatively charged components of cells eg nucleic acid and many proteins, whereas acidic dyes are repelled by them. Common basic dyes include: methylene blue, crystal violet, safranin and malachite green. Acidic dyes can find application in ‘negative staining’, where the dyes are used to stain the backgrounds against which colorless cells are seen. Examples of acidic dyes include eosin, acidic fuchsin

43. Smear preparation and fixation Smear preparation Smear is a film of dried cells. To stain a microorganism, either a drop of liquid containing the microbe or a mixture of cell colonies from solid medium and water is placed on a glass microscope slide and allowed to air dry . To give reliable information, smears must be properly prepared, labelled and fixed correctly. Smears should be spread evenly, covering an area of about 15 to 20mm on a slide Sufficient material should be applied so that chances of detection is high However, the volume of sample taken should not be so excessive, that it will interfere with the passage of light.

44. Fixation Smears are fixed to preserve the cells and its components, stops all biochemical reactions and prevent it from being washed off from the slide during staining. Smears are fixed by either alcohol or heat. Heat fixation The widely used method of fixation, however it can damage organisms and alter staining characteristics if excessive heat is used. It can also damage leucocytes and is therefore not suitable for fixing smears which may contain intracellular organisms eg N. Gonorrhoea, N. Meningitides. Techniques in heat fixation Prepare a smear. Pass slide through the flame three times (it should still be possible to lay the slide on the back of your hands after this without feeling uncomfortably hot). Allow to cool before staining

45. Alcohol fixation Acts by reducing the solubility of protein molecules. It is less damaging to the microorganisms than heat. Cells especially pus cells are well preserved. It is recommended for Gram negative diplococci eg N. Gonorrhoea, N. Meningitides. It is more bactericidal than heat, eg M. Tuberculosis is killed by 70%v/v ethanol in sputum smears. Techniques in alcohol fixation Allow smear to air dry For Gram negative diplococci, fix with one or two drops of absolute methanol or ethanol. For other organisms, fix with one or two drops of 70%v/v methanol or ethanol. Leave ethanol on smear for a minimum of two minutes or until alcohol evaporates.

46. STAINING This is a technique used to enhance contrast in microscopic image by highlighting structures for viewing. Precautions to take when staining smears Use a staining rack Do not stain a smear that is too thick because it results in poor staining and incorrect reporting Exactly follow staining techniques Label stains and reagents clearly Use dropper bottles to dispense stains and reagents. Simple Stains This involves the use of a single basic dye to stain a microbe It is used to determine size, shape, and arrangement of the microbes being observed

47. DIFFERENTIAL STAINING Differential staining techniques are used to differentiate one group of bacteria from another. Involves more than one stain. The most frequently used differential staining is Gram staining and acid fast staining Gram stain This is the most widely used procedure for staining bacteria. The basis was developed by Hans Christian Gram ( a German) in the 1884. It is an important preliminary step in bacteria characterization, identification and classification. Is used to separate bacteria into two major groups: Gram positive and Gram negative based on fundamental biochemical and structural differences of their cell walls.

48. Basic steps in Gram staining Flood the smear with the primary stain, crystal violet. Rinse off the smear to remove excess crystal violet Flood with a dilute solution of Lugol’s or Gram’s iodine. This is a mordant that increases the affinity of cellular components for a dye. The iodine combines with the crystal violet to form a dye-iodine complex (insoluble in nature), thereby decreasing the solubility of the dye within the cell. The stained smear is rinsed again with 95% alcohol for 30 seconds. This is the decolourising agent and removes the dye-iodine complex from Gram negative but not Gram positive bacteria. A counterstain (safranin dye) is then applied to impart a contrasting colour to the now colourless Gram negative bacteria. This dye stains Gram negative as well as Gram positive bacteria but because the latter is already stained purple by crystal violet, it imparts little difference to these cells. Examine with oil immersion at 1000x magnification Reliable results can only be got when Gram stain is done properly. Important: One common mistake made is to decolorize a smear for too long a time period. Over decolorized Gram positive bacteria can appear pink if the smear is decolorized for too long.

49. Acid- fast Stain ( Ziehl-Neelsen ) This is a procedure for staining a group of organisms which do not readily take up stains . Among these are members of the genus Mycobacterium including Mycobacterium tuberculosis that cause tuberculosis . The cell wall of these organisms contain a high concentration of lipid, preventing the uptake of dyes, including those used in Gram stain. Therefore , harsh methods are required for staining these organisms . Acid fast stains are used to presumptively diagnose for Mycobacterium .

50. Procedure : Prepare a smear Flood the slide with the primary dye carbol fuchsin , a red stain Heat over boiling water to allow the primary dye enter the waxy cell wall of the organism. Rinse slide to remove the residual stain Flood with acid alcohol which is a very potent decolourizer. This step removes the carbol fuchsin from nearly all organisms. However , the few unusual organisms ( Mycobacterium) still retains the dye after the intense decolourization, hence the name, acid -fast bacteria. Methylene blue is used as a counter stain, imparting a blue colour to non-acid fast cells. Acid fast bacteria does not take up the methylene blue and therefore appears reddish-pink

51. NEGATIVE STAINING Called negative staining because it does not stain the bacterial cells directly, instead, it stains the background and glass slide. Used to study the morphological shape, size and arrangement of the bacteria cells that is difficult to stain. eg : Spirilla Or cells that are too delicate to be heat-fixed. Involves only one stain (Acidic stain) with negative charges. E.g India Ink or Nigrosin . Does not involve heat fixation of sample Due to repulsion between the negative charges of the stains and the negatively charged bacterial wall, the dye stains the background.

52. Procedure: Place a small drop of nigrosin close to one end of a clean slide . Place a slide against the drop of suspended organisms at a 45° angle and allow the drop to spread along the edge of the applied slide. Using aseptic technique, place a loopful of inoculum from the bacterial culture in the drop of nigrosin and mix . Push the slide away from the drop of suspended organisms to form a thin smear. Air-dry . Examine the slides under oil immersion . Note : Do not heat fix the slide . Result: Background:dark Cell:bright

53. SPECIAL STAINS TO OBSERVE CELL STRUCTURES Dyes can be used to stain specific structures inside or outside the cell. Endospore stain Endospores are special type of dormant cell, which are resistant to destruction and staining. Examples certain Gram positive bacteria eg Bacillus and Clostridium Spore stains are used to make the endospore readily noticeable. Heat is used to facilitate staining. Procedure Prepare smear and fix Flood with malachite green Apply gentle heat to facilitate the uptake of the dye into the endospore Rinse smear with water (only endospores will retain the malachite green ) Counterstain with safranin red The endospores appear green amid a background of pink cells.

54. Flagella stain These are appendages that provide motility for prokaryotic cells. They are usually viewed using electron microscopy because they are ordinarily too thin (0.13 micron in diameter) to be seen with the light microscope. Using light microscopes, the flagella are made thick using mordant in order to increase their thickness/diameter enough to be seen. The flagella stain employs a mordant (a colloidal suspension of tannic acid or potassium alum ) that allows the staining agent to adhere to and coat the thin flagella, effectively increasing its diameter which then makes them visible. After the mordant, stain with basic fuchsin dye and observe.

55. The staining procedure is complex and requires patience and expertise.

56. Capsule staining This is employed in bacteria, including both Gram-positive and Gram-negative, that are surrounded by an outer polysaccharide-containing viscous layer termed the glycocalyx . When the composition of this layer is tightly bound and remains attached to cells, it is referred to as a capsule. Capsules are usually composed of polysaccharides; however they may also contain polyalcohols and polyamines. Functions of capsules include protection from desiccation and adherence to surfaces and other bacteria contributing to biofilm formation.  Capsules also often play a role in pathogenicity, acting as virulence factors to protect cells from phagocytosis and/or complement-mediated killing

57. The purpose of the capsule stain is to reveal the presence of the bacterial capsule. The water-soluble capsule of some bacterial cells is often difficult to see by standard simple staining procedures or after the Gram stain. Capsule staining methods were developed to visualize capsules and yield consistent and reliable results Since capsules stain poorly, the principle of negative staining is applied. The background and the cell itself is stained, allowing the capsule to stand out.

58. Ubiquity of microorganism Meaning: Microorganisms are literally present everywhere. Bacteria are extremely common microorganisms. They are known for causing diseases like pneumonia, meningitis and toxic shock syndrome, however, only 3 percent of bacteria are actively harmful to people or animals . Human body itself has about 100 trillion bacteria with most living on the skin and inside the digestive system . Harmless bacteria on the skin protect themselves from other microbes by releasing toxic proteins . In the intestines, bacteria aid in digestion, access nutrients and hinder the growth of harmful bacteria.

59. Methods to demonstrate ubiquity include: A. Petri plate exposure to atmosphere B. Swab/swab rinse method—swab method involves the use of a sterile pre-moistened swab to isolate microorganisms from the surfaces.  In swab rinse, the tip of the cotton swab is moistened by placing it in a known volume of sterile buffer/ saline. This saline is subsequently used for determination of bacteria. C. Aseptic and non-aseptic technique involving use of sterilized vs unsterilized inoculating loop D. Aseptic inoculation of solid agar media by streak plate technique, serial dilution, E. Isolation of pure culture from mixed population. etc  

60. Aseptic techniques Are all the procedures or methods used to minimize or prevent contamination of products (in this case pharmaceutical products) and infection of human beings. It is a fundamental and important laboratory skill in the field of microbiology involving the application of strictest rules and utilization of every knowledge about micro-organisms and infection prevention to minimize risks likely to be experienced in an infection /contamination . Primary goals of aseptic technique are: to prevent harmful microorganisms from spreading and causing contamination or infection; to maximize and maintain aseptic during manufacturing.

61. Every finished product should be devoid of any form of contamination (particles, bacteria, extraneous material ). Sources of contamination of Pharmaceutical products Air (by air borne microorganism including fungi) and dust. Researcher’s or investigator’s body Laboratory bench tops Laboratory floors Unsterilized glass wares and equipments .

62. Applications of aseptic technique Production of uncontaminated, safe products . Isolation of a single / pure culture from a mixed culture In sub - culturing ie transfer of cultures from one medium to another by inoculation Prevention of infection of the investigator or researcher. Prevention of spread of laboratory microorganism in the environment especially pathogens . Maintenance of pure stock culture while transferring cultures to new media.

63. Aspects of Aseptic Techniques Use of Barriers which include gloves, masks, sterile gowns, head gears etc Washing of hands and arms. Use of disinfectants during cleaning Use of inoculating or wire loops Wire loops are made of platinum or nichrome metals which are resistant to heat, inert to cells and can be heated repeatedly to red-hot temperatures for sterilization. They are 2-4mm in diameter with capacity of 0.02ml of liquid. NB: To maintain aseptic technique therefore, inoculating loop must be sterilized through FLAMING. Flaming involves holding inoculating loop over a red hot zone of Bunsen burner for about 2-3 seconds until red hot. This is done to kill contaminating organisms before it is used for transfer.

64. Flaming the mouth of Test tubes and bottles : Flaming the mouth of test tubes creates a convection current which forces air out of the tubes and makes the tube unfavourable for the air-borne contaminants that might enter the containers. Bunsen burners : Heat generated from Bunsen burners causes an increase in temperature around the work area. This prevents/ reduces the chances of product contamination by air- borne microorganisms . Laminar air flow hoods / cabinet : This discharges clean air to the working area, provides a constant flow of air to the work area whereas the air flowing out of the hood suspends and removes contaminants introduced into the work area by personnel. Within the laminar flow hood, room or lab air is passed through a pre-filter to remove all contaminants (dusts, lint), then through the High Efficiency Particulate Air (HEPA) filter where the air is purified and then let out (flow out) over the entire work surface at a uniform velocity.

65. BEST OF LUCK