Fundamental principles of microbiology

classification of microbes, staining techniques

1. Fundamental Principles of Microbiology DEVIPRIYA P V M PHARM

2. Microbiology Microbiology is the study of living organisms that are microscopic in size Medical microbiology is the study of microscopic organism that infect man, his reaction to such infections, the way in which the disease is produced and the method for diagnosis, prevention and treatment of such infectious disease. Microorganisms can be seen only with the help of microscope. Bacteria, fungi, algae, viruses, protozoa, mycoplasms , rickettsias all are microorganisms.

3. Classification of microorganism They are classified under kingdom protista . Depending on the cellular organisation and biochemistry, the kingdom protista is divided into two groups- prokaryotes and eukaryotes .

4. Character Prokaryotes Eukaryotes Cytoplasm Cytoplasmic streaming Absent Present Pinocytosis Absent Present Mitochondria Absent Present Lysosomes Absent Present Golgi bodies Absent Present Endoplasmic reticulum Absent Present Nucleus Nuclear membrane Absent Present Nucleolus Absent Present Deoxyribonucleoprotein Absent Present Number of chromosomes One More than one Mitotic division Absent Present Chemical variations Sterols Absent Present Muramic acid Present Absent

5. Bacteria A bacterium is a unicellular prokaryotic microorganism which does not contain chlorophyll and does not multiply by true branching. The unit of measurement used is micron (micrometer).

6. Shape of Bacteria: Bacteria are classified into varieties based on their shapes: Cocci are oval or spherical bacterial cells Bacilli are rod shaped cells Vibrios are comma shaped curved rods. These bacteria possess characteristic vibratory motility Spirilla are rigid spiral shaped bacteria Spirochaetes are spiral shaped cells look like coiled hairs Actinomycetes are branching filamentous bacteria. In the tissue lesions they appear as if radiating rays. Mycoplasmas are bacteria that do not have cell wall and therefore their morphology is not stable. They occur as round or oval bodies and as filaments

7. Bacteria loose their cell wall and hence their shape can change, due to defective formation of cell wall either spontaneously or as a result of administration of drugs such as penicillin. Such cells are called protoplasts, spheroplasts or L forms Arrangement of Bacteria: Cocci are arrange in pairs( diplococci ), in chains (streptococci), groups of four(tetrads) or eight( sarcina ) or as bunch of grapes (staphylococci). Bacilli can be arrange in chains (Anthrax bacilli) in cluster, in groups of two( diplobacilli eg : pneumoniae ) and sometimes at angles to each other making a cuneiform pattern( corynebacteria ).

8. Anatomy of Bacteria: It consists of an outer layer cell envelop which is differentiated into an outer rigid cell wall and beneath it a cytoplasmic membrane which is also called plasma membrane. Inside the cell envelop lies the protoplasm which contains cytoplasm and cytoplasmic inclusions such as ribosomes , mesosomes , granules, vacuoles and the nuclear body. Sometimes a bacteria is enclosed into a viscid layed which may be a loose layer or organised as a capsule. Some bacteria also posses filamentous appendages at their surface. These appendages can be flagella (organ of locomotion) and fimbriae (organ of adhesion) Bacterial nucleus does not contain nuclear membrane or nucleolus. The bacterial chromosome is haploid and replicate by simple fission

9. Spores: Some bacteria(Bacilli and clostridium) have the ability to form highly resistant resting stages called spores. a single spore is formed by one bacterium and one spore gives a single bacterial cell germination. Spores which are formed inside the bacterial cells are called as endospores . Bacterial spores are resistant to chemicals and heat. Spores of all medically important bacteria are destroyed by autoclaving at 120°C for 15 minutes. Sporulation helps the bacteria to survive for long period under unfavourable conditions. Spores germinate into bacterial cells when transferred to favourable conditions

10. It is oval or spherical in shape and is attached to the parent cell It consists of a nuclear body, surrounded by spore wall in the form of a delicate membrane. Outer to the spore wall lies a thick spore cortex , which in turn is enclosed in a multilayered tough spore coat. Spores of some bacteria have an additional outer covering called exosporium which have ridges and grooves .

11. Name of Bacteria Disease produced Staphylococci Localised suppurative lesions Stretococci Pyrogenic infections, rheumatic fever Pneumococci Pneumonia, otitis media, bronchitis and meningitis Corynebacterium diphteriae Diphtheria Bacillus anthracis Anthrax Clostridium tetani Tetanus E.Coli Urinary tract infections, gastroenteritis Shigella Bacilllary dysentry Salmonella Typhoid Vibriocholerae Cholera Pseudomonas aeruginosa Suppurative otitis , infections of wounds, bed sores etc Mycobacterium tuberculosis Tuberculosis Haemophilus influenza Influenza Mycobacterium leprae Leprosy Mycoplasma pneumonae Pneumonia Actinomycetes Actinomycosis Rickettsiaceae Typhus fever

12. Fungi Fungi are eukaryotic protists . They have rigid cell wall containing polysaccharides - chitin, mannan and others. The cytoplasmic membrane of fungi contains sterols. The nuclei contain nuclear membrane and paired chromosomes. Fungi multiply sexually, asexually or both. They can be unicellular or multicellular . Elongation of the fungi cells produces a thin tubular structure called hypha . A tangled mass of hyphae is called mycelium Fungi which can make mycelia are called filamentous fungi or moulds

13. Types of fungi : Fungi are divided into 4 on the basis of cell morphology. Yeasts: Are unicellular fungi Their cells are spherical or ellisoidal in shape. They multiply by budding. The pathogenic yeast is crytococcus neoformans . Yeast like fungi: They partly grow as fungi and partly as elongated cells like hyphae called pseudomycelium Eg : candida albicans Moulds / filamentous fungi: Can produce true mycelia Multiply by spore formation Eg : micosporum , trichophyton , epidermophyton

14. Dimorphic fungi: They occur as filaments or as yeasts. In tissues or in culture at 37°C they occur as yeast, while in the soil and in culture at 22°Cthey appear as moulds. Eg : Blastomyces dermatitidis , Histoplasma capsulatum etc

15. Systematic classification of fungi: The systematic classification is based on the sexual spore formation. Lower fungi : Phycomycetes Higher fungi: Ascomycetes , Basidiomycetes Fungi imperfecti ( Deuteromycetes or Hypomycetes ) Phycomycetes : They are lower fungi These fungi have non segmented hyphae. They form endogenous asexual spores called sporangiospores which are contained in a sac like structure called sporangia Some of them produce sexual spores called Oospores

16. Higher fungi: These fungi have separated hyphae They form exogenous asexual spores called Conidia . Ascomycetes : These fungi form sexual spores called ascospores within a sac or ascus Yeast and filamentous fungi are included in this class Basidiomycetes : These fungi form sexual spores known as basidiospores on a basidium or base. Fungi imperfecti : Sexual phase have not been identified. Fungi of medical importance

17. Comparison of Fungi and Bacteria Sl no Characters Fungi Bacteria 1 Diameter 4 μ 1 μ 2 Nucleus Eukaryotic Prokaryotic 3 Cytoplasm Present Absent 4 Cell membrane Sterols present Sterols absent 5 Cell wall content Chitin(polysaccharide) peptidoglycan 6 Spores Sexual and asexual spores for reproduction Endospores for survival and not for reproduction 7 Thermal dimorphism Present in some Absent

18. Laboratory Diagnosis of Fungi: The diagnosis is made by morphological study of the materials taken from the lesions. Skin scrapings are treated with 10% potassium hydroxide and then examined under microscope as wet mount. For microscopic study, a small bit of fungus colony is taken on a slide and mounted in lactophenol cotton blue. Slide culture technique is used for morphological study of fungus The periodic acid schiff (PAS) and methanamine silver strains are valuable methods for the demonstration of fungal elements in tissue sections

19. Culture of fungus: The common culture media of fungi are: sabouraud’s glucose agar Czapek – Dox medium Cornmeal agar To prevent bacterial contamination, antibiotics are added in the media. Cycloheximide incorporation in the medium inhibits many contaminant moulds Cultures are incubated at room temperature (22°C) for weeks or at 37°C for days.

20. Growth characters: Growth characters useful for identification are the rapidity of growth, morphology as well as the color of pigmentation of the colony. The diameter of hyphae, presence or absence of septa and other special structures are also helpful in the diagnosis Spiral hyphae : they are spring like helical coils Racquet hyphae : these localised swellings are formed by twisted hyphae and look like tennis racquets. Favic chandlier hyphae : contain numerous long branches at the end of hyphae.

21. The morphology of asexual spores is also of diagnostic value. Microconidia are small single shaped spores Macroconidia are large, single or multicellular spore Blastospores are formed by budding as in yeast Arthospores are formed along the mycelium by segmentation and condensation of hyphae Chlamydospores are thick walled resting spores formed by rounding up and thickening of the segments of the hyphae. Fungal infection (mycoses): 4 types: Superficial, Sub cutaneous, Deep seated (systematic) Opportunistic

22. Superficial fungal infections: The fungi causing the disease are specialised saprohytes which can digest keratin of the skin including nails & hairs. It include various types of tinea and ringworm affecting the skin, hair and nails. Causative fungi for superficial infections are dermatophytes , candida albicans , pityrosporum orbiculare , cladosporium , piedraia hortai . Subcutaneous fungal infections(Subcutaneous mycosis): Caused by fungi such as rhinospordium , fonsecaea , madurella norcardia , sporotrichum etc.

23. Deep seated fungal infections(systematic mycosis): Are caused by soil saprophytes . severity ranges from asymptomatic infections to fatal diseases The causative agents include actinomycetes (produces madura foot), rhinosporidium seeberi (produce rhinosporidiosis ), cryptococcus neoformans ( cryptococcosis ), dimorphic fungi- blastomyces dermatitidis ( blastomycoses ) and histoplasma capsulatum (produce histoplasmoses ) Opportunistic fungal infections: Occur in patients suffering from diseases such as cancer, AIDS or diabetes. The use of drugs like immunosuppresive agents, steroids, board spectrum antibiotics and exposure to X-rays makes the body vulnerable to these infections. Caused by mucor , penicillium , rhizopus and aspergillus

24. Mycotic poisoning: Many fungi can produce poisoning. Are of 2 types: mycetism and mycotoxicosis Mycetism : Fungi which is eaten for itself and produces toxic effects. Eg : some mushrooms after ingestion causes toxic effects such as GIT disturbances, dermatitis or even death. Mycotoxicosis : The toxins produced by fungi ie , mycotoxins contaminate the food material and produce side effects. Eg : aflatoxin produced by Aspergillus flavus present in mouldy foods such as ground nuts, corn and peas can produce carcinogenic effects in humans Ergotism : Poisoning caused by eating rye, infected with fungus ergot.. Symptoms include gangrene of fingers & toe with diarrhea and vomitting , nausea and headache

25. Viruses These microorganisms are much smaller than bacteria . They do not have a cellular organisation. They contain only one type of nucleic acid , either DNA or RNA. They do not contain enzymes necessary for protein and nucleic acid synthesis. Viruses are dependent on the host cell for replication . They are obligate intracellular parasites that are not affected by the usual antibiotics. The extracellular infectious virus particle is called virion

26. Structure of virus

27. Viruses produce diseases like AIDS, cancer, rabies or yellow fever etc. The viral diseases can be sporadic like mumps, endemic such as measles or pandemic such as influenza. Size: Viruses vary in size The largest virus such as pox virus is 300 nm in size. The smallest viruses such as those causing foot and mouth diseases are 20 nm in size . Shape and Structure: The viruses vary in shape. Most viruses are spherical. The rabies virus is bullet shaped, the pox virus is brick shaped and the tobacco virus is rod shaped. Bacterial viruses have a complex morphology

28. Virion: Virion consists of nucleic acid. Core surrounded by a protein covering called capsule. The capsule together with the nucleic acid is called nucleocapsid . The capsid is formed by a large number of capsomers made up of polypeptide molecules. The main function of capsid is to introduce viral genome into the host cells by adsorbing readily to the cell surfaces. Two types of symmetry are seen in the capsid , ie , icosachedral (cubical) and helical Virions may be enveloped or non-enveloped (naked). The envelop or outer covering of the viruses is derived from the host cell membrane at the time of budding of virus and consists of lipoprotein.

29. The protein subunits projecting likes spikes on the surface of the envelope are called peplomer . A virus may have more than one peplomer . Envelopes confer chemical, antigenic and biological properties on viruses.

30. Classification of viruses: Classified into two based on the presence of nucleic acid present. (1) The Deoxyriboviruses or DNA viruses: These viruses contain DNA in them. Eg : poxvirus, Herpes virus, Adenovirus, Papilloma virus, Human Hepatitis Type B virus etc (2) The Riboviruses or RNA viruses: These viruses contain RNA in them. Include Enteroviruses , Rhinovirus, Influenza virus, Mumps virus, Cholera virus, Rabies virus, Corona virus, Retrovirus etc

31. DNA virus RNA virus

32. Isolation of microorganisms For a proper diagnosis of infectious diseases , it is necessary to know the type and nature of causative microorganism. For this the specimen of the infected material are collected. Mostly used specimens are swabs, pus, sputum, urine, stool , blood, cerebrospinal fluids, pleural fluids and aspiration material. The specimen should be collected before starting the antimicrobial treatment otherwise the specimen may become sterile. The specimen should be collected from the site most likely to be infected by the microorganism

33. Eg : sputum is examined in respiratory tract infections, stool in diarrhoea , cerebrospinal fluid in meningitis and blood in enteric fever is studied for the presence of microbes. Stage of disease is an important factor for specimen collection. Eg : in the early stage of enteric fever blood culture is done, in the second week widal test is done and in third week stool culture is performed. Timing of collection is also important for the successful isolation of causative microorganism Eg : In UTI the first morning sample of urine is best for culture.Sputum and conjunctival swabs should be collected in the morning

34. The specimen should be collected in sufficient quantity in suitable containers. Urine sample is collected in sterile test tube. Swab from eye, throat, rectum or vagina should be collected by sterile swab stick and that too should be placed into sterilized test tube immediately after taking the sample. Sputum should be collected in petridish. Cerebrospinal fluid is collected in sterilized vials. Blood for culture is collected in blood culture bottles. After collection the specimen should be delivered to laboratory without any dela y to avoid the overgrowth of organisms. Sufficient clinical information should always be given with the specimen

35. Methods of isolating pure cultures Surface planting method: the distinct colonies of bacteria are picked out for further purification and examination Enriched selective and indicator media are used widely for isolation of bacteria from specimens. Pretreatment of specimens with appropriate bactericidal substances destroys the unwanted bacteria and pure culture can be obtained. Tubercle bacilli are isolated by this method from sputum Obligate aerobes and anaerobes can be separated by cultivating the specimen in aerobic and anaerobic conditions

36. Separation of bacteria with different optimum temperatures can be done by incubating the material at different temperatures. Thermophilic bacteria grow at 60°C and Neisseria catarrhalis can be grown at 22°C. By heating at 80°C the vegetative bacteria can be eliminate from the mixture of vegetative and spore forming bacteria. Motile and non-motile bacteria can be separated by using the Craigie’s tube- a tube containing semisolid agar. By inoculation into appropriate animals the pathogenic bacteria are isolated from a mixture, for example anthrax bacilli are inoculated into guinea pig or mice. By using selective filters, bacteria of different sizes may be separated. By single cell method using micromanipulators, a single bacterium is separated and cultured

37. Staining techniques After the isolation of causative microorganism, the morphological detail is studied. For morphological study the bacteria are stained properly. By staining the bacteria become clearly visible and can be identified. Smear preparation: First step in staining procedure. A loopful of liquid culture or fluid specimen or a section of bacterial colony is taken and spread as a thin film over the slide. The smear is dried in air and heat fixed by passing through a flame gently.

38. Simple staining of bacteria: Apply the stain to be used over the prepared smear. Crystal violet, methylene blue, fuchsin or safranin . Allow the stain to react for 30 seconds to 3 minutes depending on the stain used. Wash the smear with gentle stream of cold water. Dry between bibulous paper and examine under oil immersion lens of the compound microscope. Special staining procedures: Gram’s staining methods The most important staining method for bacteria The shape, size and structural details of microorganisms are made visible. Helps to group organisms into gram positive and gram negative

39. Steps involved: Prepare a thin film and dry it. Stain the prepared smear with methyl violet for one minute. Wash off the excess stain with Gram’s iodine solution. Cover the whole slide with fresh iodine solution for one minute. The smear is then decolorised with spirit. Wash the smear quickly with running tap water. Cover the smear with dilute carbol fuchsin for 30 seconds. Wash it with tap water and then dry it in air Examine the slide under oil immersion lens

40. Gram positive bacteria retain the violet colour of methyl violet. Gram negative bacteria are decolorized by spirit, alcohol or acetone and are stained with a counter stain like carbol fuchsin which imparts a pink color to them. Examples for Gram positive bacteria : Staphylococci, Corynebacterium diphtheria, Bacillus anthracis , Clostridium tetani , Clostridium welchi , Streptococci, Pneumonococci etc Examples for Gram negative bacteria : Gonococci, Meningococci , E.coli , salmonella typhi etc

41. Acid Fast Staining : The acid fast stain is a differential stain used to identify acid fast organism such as members of the genus Mycobacterium. The technique was discovered by Ehrlich who observed that after staining with aniline dyes, tubercle bacilli resist decolourisation with acids. Ziehl Neelsen method : Prepare a smear of the mucoid part of the sputum on a slide and fix it. Put strong carbol fuchsin over the smear for five minutes. Wash the smear with water. Put 20% sulphuric acid for one minute. Wash the slide again with water.

42. Now put methylene blue for 30 seconds. Wash the slide with water. Dry the slide and observe it under oil immersion lens. The pink coloured , rod shaped tubercle bacilli will be seen scattered in the sputum of open cases of tuberculosis. The acid fast organism are stained pink or bright red. These organisms after being stained with carbol fuchsin do not loose their red colour when washed with acids Staining of Spirochaetes : Spirochaetes are stained by fontana method. Ammonia silver nitrate stain is used which increases the apparent dimensions of the spirochetes and these microorganisms are seen as opaque black spiral hairs against a light back ground

43. Staining of Rickettsiae : Rickettsiae are gram negative but they are not stained well wiith gram’s stain. They are stained with Giemsa or casteneda method Staining of Yeast and Fungi: Fixed smear of yeast can be stained with crystal violet or methylene blue. These dyes are put for 30 seconds to one minute. Wet mounts of yeast can be stained effectively with methylene blue or Gram’s iodine. The cell can be emulsified in a drop of the either stain and covered with a cover glass. Lactophenol cotton blue is excellent for staining fungi.