Lec 01. introduction to microbiology

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  • Introduction to Microbiology

    Sebghatullah Mansoor

    BS (Medical Sciences), MS (Microbiology), MPH (Continue)

    Medical faculty or Malalay University


  • Definition Microbiology is the study of microorganisms, a large and diverse

    group of microscopic organisms that exist as single cells or cell clusters;






    Environment effects

    Relationship with other organism or Human being


    The study of small organisms.



    Viruses, Viroids & Prions(not really organisms)


    Algae Fungi Protozoa

  • Microorganisms have a remarkable impact on all life and the physical and chemical makeup of our planet.

    They are responsible for cycling the chemical elements essential for life, including carbon, nitrogen, sulfur, hydrogen, and oxygen

    there are 100 million times as many bacteria in the oceans as there are stars in the known universe.

    More than 90% of the cells in our bodies are microbes

    The bacteria present in the average human gut weigh about 1 kg, and a human adult will excrete his or her own weight in fecal bacteria each year


  • At the beginning the bacteriology were termed for microbiology,

    By discovery of microscope (Robert Hookes Antonie van Leeuwenhoek), A hug evolution occurred in the history of medical science, specially in the microbiology science which by understanding other infectious agents such as Prions, Virus , Rickettsia , Fungus & Protozoa, the Bacteriology named were changed to Microbiology


  • Generally Microbiology is classified as follow;

    Medical Microbiology

    Industrial Microbiology

    Food Microbiology

    Soil Microbiology

    Marine Microbiology

    Plant Microbiology


  • Medical Microbiology Medical microbiology is a branch of medical science concerned with the

    prevention, diagnosis and treatment of infectious diseases. In addition, this field of science studies various clinical applications of microbes for the improvement of health, and classify as follow ;







  • Golden age of Microbiology

  • There are many methods to classify micro-organisms, which include;

    Binomial classification

    Classification according to Temperature

    Classification according to Gram staining

    Classification according to Presence of Oxygen

    Classification according to Morphology of bacteria

    Classification of Microorganisms (Taxonomy)

  • Classification of Microorganisms (Taxonomy)

    Binomial classification KINDOM the highest level in classification

    PHYLUM related classes

    CLASS related orders

    ORDER related families

    FAMILY related genera

    GENUS closely related species

    SPECIES organisms sharing a set of biological traits and reproducing only with their exact kind

    Further classifications especially with bacteria:

    Strainorganisms within a species varying in a given quality

    Typeorganisms within a species varying immunologically

  • Classification of Microorganisms (Taxonomy)Kingdom: Eubacteria

    Phylum: Proteobacteria

    Class: Gammaproteobacteria

    Order: Enterobacteriales

    Family: Enterobacteriaceae

    Genus: Escherichia

    Species: E. coli

    For example ;

    Escherichia coli

  • Classification according to Temperature Psychrophiles (Philic) ; Can survive under 15-25 C . e.g; Bacillus psychrophilus Mesophiles ; Can survive under 25-45 C . e.g; E.coli Thermophiles ; Can survive under 45-60 C . e.g ; Bacillus stearothermophilus

    Classification according to Gram Staining Gram Positive Bacteria, e.g; Staphylococcus aureus Gram Negative Bacteria, e.g ; E. coli

    Classification according to Presence of Oxygen Obligate aerobic; Which Oxygen is the primary needs. e,g; Mycobacterium tuberculosis Factitive anaerobic; Can survive with or without oxygen. e,g; E.coli Anaerobic; Which can not survive in the presence of Oxygen. e,g: Clostridium tetani Microaerophilic; Which need less amount of Oxygen. e,g ; Neisseria gonorrhea Aerotolerant; Do not required oxygen , nether dies in the presence of oxygen.

    e,g ; Lactobacillus

  • Classification according to Morphology

    Cocci ; Round shape , Diplo cocci ( two cocci), staphylococci ( cluster of cocci), Streptococci (chain of cocci)

    Bacilli ; Rod shape Vibrios ; Comma shape Spirilla ; Flexible spiral shape Spirochetes ; Spring type shape Actinomycetes ; Branching filamentous bacteria Mycoplasma ; Cell wall less bacteria

  • Classification according to Gram Staining

  • 1. Obligate aerobic2. Anaerobic3. Factitive anaerobic4. Microaerophilic5. Aerotolerant

    Classification according to Presence of Oxygen

  • Classification according to Morphology

  • Classification according to Morphology

  • Classification according to Morphology


  • Generally all organism are classified as follow

    Eukaryotes;-- Which has a true nucleus & all cellular organelles e,g: Fungi, Protozoa, Algae..

    Prokaryotes;-- Which has no true nucleus and missing some of cellular organelles e,g: Bacteria

    Viruses; -- Which is composed of Capsid or some have outer protein coating called envelop, and DNA or RNA not both. .e.g ; Polio virus, Hepatitis viruses

  • Virus

  • Characteristics of Prokaryotic and Eukaryotic Cells

  • Structure of bacteria Bacteria are classified by shape into three basic groups: cocci,

    bacilli, and spirochetes.

    The cocci are round, the bacilli are rods, and the spirochetes are spiral-shaped.

    Some bacteria are variable in shape and are said to be pleomorphic (many-shaped).

    The shape of a bacterium is determined by its rigid cell wall.

    The microscopic appearance of a bacterium is one of the most important criteria used in its identification.

  • A typical structure of bacteria

  • The major components of bacterial cells are;

    Cell wall

    Cytoplasmic membrane




    Structure of bacteria

  • Cell wall

    All bacteria have a cell wall composed of peptidoglycan except Mycoplasma,which are surrounded only by a cell membrane.

    The concept of gram positive & gram negative bacteria is based on bacterial cell wall which gram negative bacteria have a thin peptidoglycan covered by an outer lipid-containing membrane, whereas gram-positive bacteria have a thick peptidoglycan and no outer membrane.

    These differences explain why gram-negative bacteria lose the stain when exposed to a lipid solvent in the Gram stain process, whereas gram-positive bacteria retain the stain and remain purple.

    For better understanding we must to know about Gram staining method

    Structure of bacteria

  • Staining

    Micro-organism can not be seen by naked eyes, that why we must magnify the size of micro-organism For that reason we need a magnifier which is microscope which enlarges the organisms almost 1000 more than its actual size.

    The size of bacteria is measures by micron meter (m)

    The size of most bacteria rages from 1 to 3 m.

    The smallest bacteria is Mycoplasma 0.2 m, where as the largest is Borrelia 10 m

  • Most of the micro-organism are enable to seen even by microscope, we need to stain the organism

    According to PH all stains are acidic, alkali or neutral. Generally acidic materials are stains with alkali or in contrast alkali materials

    are stains with acidic . In general we classify the staining methods as follow. Simple staining Differential staining ( Gram staining) Acid fast staining Negative staining Flagella staining Capsule staining Spore staining


  • Simple staining

    in simple staining we use a single dye (methylene blue, carbol fuchsin, Safranin ) here just observe the shape of organism or bacteria either its cocci, rods or spirochetes


    1. Make a thin smear on a glass slide of a given sample

    2. Air dry it and cover with Methylene blue (CF, S)

    3. Wash with tap water and again air dry it

    4. Observe under the microscope , if the given sample is bacteria then we will see Bacilli or Cocci shapes of bacteria


  • Safranin stained bacilli Methylene Blue stained cocci

  • Differential staining (Gram Staining)

    Gram stain is the most important staining procedure which differentiate all the bacteria in two main categories either gram negative or gram positive bacteria. This method was discovered by Hans Christian Gram in Germany in 1884

    In this staining method we use more then one type of dye which include

    Crystal violet


    The mechanism of gram positive & gram negative bacteria is based on bacterial cell wall which gram negative bacteria have a thin peptidoglycan covered by an outer lipid-containing membrane, whereas gram-positive bacteria have a thick peptidoglycan and no outer membrane.


  • Gram positive Gram negative

  • Gram stain


    1. Make a smear of bacterial specimen on a glass slide

    2. Air dry it an cover with crystal violet for 2-3 minutes

    3. Wash with tap water and cover with grams iodine for 2 minutes

    4. Wash with tap water and cover with grams alcohol (95%) for 30 seconds to 1 minute

    5. Wash with tap water and cover with Safranin for 2 minutes

    6. Wash with tap water and observed under the microscope which will results either purple or pink, red colored bacteria

    7. The Purple colored bacteria is named gram positive bacteria & the Pink or red colored bacteria named gram negative bacteria


  • Gram stain

    2-3 m 2 m 30 sec-1 m 2m

  • Gram stain

  • Acid fast staining (ZiehlNeelsen stain) Ziehl-Neelsen stain was discovered by the bacteriologist Franz

    Ziehl (18591926) and the pathologist Friedrich Neelsen (18541898).

    It is a special bacteriological stain used to identify acid-fast organisms,

    The mycobacterium tuberculosis is deferent from other bacteria in the composition of cell wall which in addition to peptidoglycan, the outer membrane or envelope of the acid-fast cell wall of contains large amounts of glycolipids, especially mycolic acids that in the genus Mycobacterium, make up approximately 60% of the acid-fast cell wall.

  • Structure of Mycobacterium tuberculosis

  • Procedure

    1. Make a smear of sputum on glass slide and fixe with ethanol & air dry it

    2. Cover slide with Carbol fuchsin and heated for 5 minutes

    3. Wash with tap water & cover with acid alcohol for 1-2 minutes

    4. Wash with tap water & cover the slide with Methylene blue for 2 minutes

    5. Wash the slide and observed the red rods under microscope which will be mycobacterium tuberculosis

    Acid fast staining

  • Acid fast staining

  • TB slide after AFB stain

    The arrow shows red rods bacilli

  • Negative staining (India Ink )

    Negative staining is a recognized method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid.

    In this technique, the background is stained, leaving the actual specimen untouched, and thus visible


    1. Make a smear from a given sample & mix with Negrosin or Congred dye

    2. Air dry it and observed under Bright field microscope


    The background will take the color but the bacteria will not , which the bacteria will appear bright

    Note: Mostly used for Spirilla or for electron microscopy

  • Negative staining

    A & B are the colorless bright bacteria

    C shows the staining method


  • Flagella stain

    The flagella stain allows observation of bacterial flagella under the light microscope. Bacterial flagella are normally too thin to be seen

    Therefore the flagellated bacteria are stained with special dye is commercially available.

  • Capsule staining

    A capsule is a gelatinous outer layer secreted by bacterial cell and that surrounds and adheres to the cell wall

    The main purpose of capsule stain is to distinguish capsular material from the bacterial cell.

    Most capsules are composed of polysaccharides, but some are composed of polypeptides.

    The capsule stain employs an acidic stain and a basic stain to detect capsule production

  • Procedure;1. Place a small drop of a negative stain (India Ink, Congo Red, Nigrosin, or Eosin) on

    the slide2. Add a loopful of bacterial culture to slide, smearing it in the dye3. Use the other slide to drag the ink-cell mixture into a thin film along the first slide

    and let stand for 5-7 minutes4. Allow to air dry (do not heat fix)5. Flood the smear with crystal violet stain (this will stain the cells but not the

    capsules) for about 1 minutes.6. Cover the slide with copper sulfate (20%)7. Drain the crystal violet by tilting the slide at a 45 degree angle and let stain run off

    until it air dries .8. Examine the smear microscopically (100X) for the presence of encapsulated cells

    as indicated by clear zones surrounding the cells.

    Capsule staining

  • Capsule staining

  • Spore staining

    The endospore stain is a differential stain used to visualize bacterial endospores.

    Endospores are formed by a few genera of bacteria, such as Bacillus . By forming spores, bacteria can survive in hostile conditions.

    Spores are resistant to heat, dryness, chemicals, and radiation

  • Procedure;

    1. Prepare smears of organisms to be tested for presence of endospores on a clean microscope slide and air dry it.

    2. Heat fix the smear.

    3. Place a small piece of blotting paper (absorbent paper) over the smear and place the slide (smear side up) on a wire gauze on a ring stand.

    4. Heat the slide gently till it starts to evaporate (either by putting the slide on a staining rack that has been placed over a boiling water bath or via bunsen burner).

    5. Remove the heat and reheat the slide as needed to keep the slide steaming for about 3-5 minutes. As the paper begins to dry add a drop or two of malachite green to keep it moist, but dont add so much at one time that the temperature is appreciably reduced.

    6. Remove the blotting paper and allow the slide to cool to room temperature for 2 minutes.

    7. Wash the slide with tap water (to wash the malachite green from both sides of the microscope slide).

    8. Stain the smear with safranin for 2 minutes.

    9. Wash both side of the slide to remove the secondary stain and blot the slide/ air dry.

    Spore staining

  • Spore staining

  • The vegetative forms are pink color

    The spores forms are greenish blue color

    Spore staining

  • Protoplasts

    A plant, bacterial or fungal cell that had its cell wall completely or partially removed using either mechanical or enzymatic means. A further differentiation can be made for bacteria: protoplasts: is that a gram positive bacteria have their cell wall entirely removed by enzymatic means (which form L-shapedwhich will survive & return in suitable condition) or other external factors or the bacteria place in such a place which the osmotic pressure is higher then the bacterial inner osmotic pressure, in this case the bacteria will lysis.

    If the outer and inter osmatic pressure is same then the bacteria will not lysis, this process is called protoplasts

  • Protoplasts

  • Protoplasts

  • Spheroplasts If a gram negative bacteria have their cell wall entirely removed by enzymatic

    means or other external factors or the bacteria place in such a...