Microbiology Laboratory. The methods of microbiology investigation. Microscopic methodGumral Alakbarova, MD, PhD

Definitions

• Microbiology from Greek μῑκρος, mīkros, "small"; βίος, bios, "life"; and -λογία, -logia• Medical microbiology • is a branch of medical science • concerned with the prevention, diagnosis and

treatment of infectious diseases • There are four kinds of microorganisms that cause

infectious disease:• bacteria, • fungi,• parasites • viruses

History of Microbiology

• 1673-1723, Antoni van Leeuwenhoek (Dutch) described live microorganisms that he observed in teeth scrapings, rain water, and peppercorn infusions

History of microbiology

• Anton van Leeuwenhoek (1632–1723): was the first microbiologist and the first person to observe bacteria using a single-lens microscope of his own design.• Louis Pasteur (1822–1895): Pasteur developed a process (today known as

pasteurization) to kill microbes. pasteurization is accomplished by heating liquids to 63 to 65 C for 30 minutes or to 73 to 75 C for 15 seconds.• Robert Koch (1843–1910): was a pioneer in medical microbiology and

worked in cholera, anthrax and tuberculosis. He was awarded a Nobel prize in 1905 (Koch's postulates) he set out criteria to test.• Alexander Fleming (1929): Discovered penicillin.

Definitions

Medical microbiology laboratory

• a place when the diagnosis of infectious microorganisms takes place

• identification of the best treatment options for infection

• and the monitoring of antibiotic resistance

It also includes testing for how well a patient is responding to treatment of infection

Medical microbiology laboratories

• The Clinical Microbiology Laboratory is a full laboratory including• Bacteriology,• Mycology, • Parasitology,• Virology• Immunology• Molecular Microbiology laboratories

Bacteriology Laboratory

• isolates and identifies clinically significant microorganisms from clinical specimens • performs antimicrobial susceptibility testing

on these bacterial pathogens• Work steps of laboratory• Reception of sample • Sample cultivation • identification of bacteria• Antibiotic sensitivity testing

Mycology Laboratory

• performs identification and anti-fungal susceptibility testing on clinically significant yeast isolates• provides identification of

pathogenic moulds recovered from clinical specimens

Parasitology Laboratory• Provides services for the diagnosis of various parasitic infections

Virology Laboratory

• Provides services to aid in the diagnosis of viral infections

Serology Laboratory

• Analyzes blood specimens for diseases of public health significance• A serology blood test is performed to detect and measure the levels

of antibodies as a result of exposure to a particular bacteria or virus

Molecular Microbiology

• examines the origins of disease at the molecular level, primarily by studying nucleic acids• Deoxyribonucleic acid (DNA), which contains the blueprint for

constructing a living organism, is the centerpiece for research and clinical analysis

Microbiology Lab Tools And

Equipment

• Light microscope • Incubator • Oven • Autoclave • Fridge and refrigerator • Hood (safety cabinet) • Electronic balance

• pH meter • Hot-plate • Electronic micropipette • Water bath • Distillator • Flasks, beakers• Petri-dishes• loops • Vortex mixer • Centrifuge

Microbiology Lab Tools And Equipment

Colony counter

Magnetic stirrer

Spectrophotometer

High Performance Liquid Chromatography (HPLC)

Gas Chromatography (GC)

Gel Electrophoresis

VITEK 2

BacT/ALERT 3D system

Polymerase Chain Reaction

Microscopy

• What is a microscope? • A microscope derived from the Greek word

‘micron’= small and ‘scopos’= to look/

• Is an instrument used to see objects that are too small to be seen by the naked eye• It is an optical instrument consisting of a combination

of lenses which magnifies the image of the object seen through it• Micro= small scope=to view • Types of microscopes • Simple • Compound • Electron

Historical Background

• 1590— Hans Janssen and his son Zacharias Janssen, invented the first compound microscope

Historical Background

• 1609—Galileo Galilei developed a compound microscope and he called it the "occhiolino" or "little eye." • His first microscopes, in 1609, were basically little telescopes

with the same two lenses: a bi-convex objective and a bi-concave eyepiece.

Historical Background

•1620— Christian Huygens, developed a simple 2-lens ocular system that was chromatically corrected

Historical Background

• ANTON VAN LEEUWENHOEK – He is commonly known as “THE FATHER OF MICROBIOLOGY", and considered to be the first microbiologist• He is best known for his work on the improvement of the microscope

and for his contributions towards the establishment of microbiology• He discovered bacteria, free-living and parasitic microscopic protists,

sperm cells, blood cells, microscopic nematodes, etc.

Microscope

• Main parts of microscope:• ocular lens,• objective lens,• arm, • stage, • stage clips, • coarse adjustment knob • fine adjustment knob,• light source,• condenser • base

Microscope parts

• Base• used in carrying the microscope; houses the light source

• Arm • along with base, used as a carrying handle for the microscope

• Light source• provides illumination to specimen

• Stage• platform which the specimen rests on

Microscope parts• Coarse adjustment knob • controls the distance between the specimen and the stage; used

with scan and low power objective lenses• Fine adjustment knob• controls the distance between the specimen and the stage; used

with high dry and oil immersion objective lenses•Mechanical stage knobs• allows the movement of the stage from left to right and forward to

backward

Microscope parts

• Condenser• collects and concentrates light emitted from the light sources into a tight

beam before passing it through the specimen• Iris diaphragm• located within the condenser; tightens or widens the beam of light emitted

from the condenser

• Light intensity adjuster• controls the amount of light emitted from the source bulb

Microscope parts

• Objective lenses• serve to magnify the specimen in part (4X, 10X, 40X, 100X)

• Revolving nosepiece• allows the parfocal exchange between objective lenses during specimen

magnification• Ocular lens• allows the image to be viewed; magnifies the specimen 10X; may be

monocular or bioncular

Microscopy

• In medical microbiology, it is used in detection of micro- organisms that prepared as smear for clinical sample (i.e. gram stain results, Ziehl-Neelsen stain result). • Ocular lens with constant magnification (10X)• Objective lenses could be (4X, 10X, 40X, 100X)• After each use, microscope maintenance is obligatory

Light Microscope Electron Microscope

Differences between Light Microscope and Electron MicroscopeLight Microscope Electron Microscope

Illuminating source is the Light. Illuminating source is the beam of electrons.

Specimen preparation takes usually few minutes to hours. Specimen preparation takes usually takes few days.

Live or Dead specimen may be seen. Only Dead or Dried specimens are seen.Condenser, Objective and eye piece lenses are made up of glasses. All lenses are electromagnetic.

It has low resolving power (0.25µm to 0.3µm). It has high resolving power (0.001µm), about 250 times higher than light microscope.

It has a magnification of of 500X to 1500X. It has a magnification of 100,000X to 300,000X.

The object is 5µm or thicker. The object is 0.1µm or thinner.Image is Colored. Image is Black and White.Vacuum is not required. Vacuum is essential for its operation.

There is no need of high voltage electricity. High voltage electric current is required (50,000 Volts and above).

There is no cooling system. It has a cooling system to take out heat generated by high electric current.

Filament is not used. Tungsten filament is used to produce electrons.

Radiation risk is absent. There is risk of radiation leakage.

Specimen is stained by colored dyes. Specimen is coated with heavy metals in order to reflect electrons.

Image is seen by eyes through ocular lens. Image is received in Zinc Sulphate Fluorescent Screen or Photographic Plate.It is used for the study of detailed gross internal structure.

It is used in the study of external surface, ultra structure of cell and very small organisms.

Incubator

• Is a device used to grow and maintain microbiological cultures • The incubator maintains optimal temperature,

humidity and other conditions such as the carbon dioxide (CO2) and oxygen content of the atmosphere inside

Autoclave

• An autoclave is a pressure chamber used to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 °C for around 15–20 minutes depending on the size of the load and the contents. • Used to sterilize culture media, discard, and

other equipments

Inoculating loops and needles• Inoculating loops are used to transfer

microorganisms to growth media or for staining slides• The wire forms a small loop with a diameter of

about 5 mm• The loop of wire at the tip may be made of

platinum or nichrome

Slide 1DefinitionsHistory of MicrobiologyHistory of microbiologyDefinitionsMedical microbiology laboratoriesBacteriology LaboratoryMycology LaboratoryParasitology LaboratoryVirology LaboratorySerology LaboratoryMolecular MicrobiologyMicrobiology Lab Tools And EquipmentMicrobiology Lab Tools And EquipmentMicroscopyHistorical BackgroundHistorical BackgroundHistorical BackgroundHistorical BackgroundSlide 20MicroscopeMicroscope partsMicroscope partsMicroscope partsMicroscope partsMicroscopyLight Microscope Electron MicroscopeSlide 28Slide 29Slide 30IncubatorAutoclaveInoculating loops and needles