Notifications
Clear all

Bacteriology practical


ug
Posts: 33
 ug
Admin
Topic starter
(@mr-right)
Reporter
Joined: 4 months ago

Microscopic size of bacteria and viruses.  

The size of bacteria is measured by the use of a graduated eye piece that is calibrated by a micrometre slide and the unit of measurement is micrometres. The micrometre is 1/1000 of a millimeter which is 0.001 millimeter. Viruses, being smaller than bacteria, are generally measured in millimicrons or millimicrometers (1/1000 of a micrometre or

0.001 micrometre or 1 millimicrometer; this is now called nanometre)

 

Bacteria

Bacteria are microscopic unicellular organisms. The smallest having a diameter of about 0.5 micrometre. The diagram on the board is a bacteria cell showing some of the essential constituents. The cell wall is a complicated lattice structure of lipoproteins, lipopolysaccharides and peptidoglycans, which give the bacteria cell its shape and also protect its cytoplasmic membrane. 

 

Morphology

Bacteria can be classified into the following type of cells: 

  1. The ovoid/ spheroid called coccus.
  2. The rod/ cylindrical called bacillus.
  3. The curved called Vibrio.
  4. The spiral shaped called spirillum.
  5. The coiled shape called spirochaetes.

The coccus (cocci) is 0.5-10 micrometres in diameter. They generally have one axis approximately equal to any other axis or distorted in some way as to depart from spherical shapes e.g. in streptococci. If after binary fission the daughter cell remains attached to the parent cell, but separates before fission occurs again, this pair of cocci are called diplococci e.g. Nessariae gonorrhoea is a gram negative streptococci. If fission continues while they remain attached forming chains, they are termed streptococci. If the division is not in one plane and random clumps of cocci occur, they are called staphylococci. The bacillus (bacilli) is 1-10 micrometre in length, 0.3 - 10 micrometre in width. The bacilli or rods do not form as many groupings as the cocci only forming diplobacilli or streptobacilli (pairs and chains). 

 

Bacteria metabolism

To obtain optimal bacterial growth in laboratory prepared media, it is necessary to understand the metabolic role of nutrients. Metabolism can be considered as an interacting set of chemical reactions of which very few occur spontaneously and most have to be catalized by specific proteins (enzymes). There are two main types of reaction: those resulting in the breaking down of molecules (catabolic reaction) and those resulting in the synthesis of molecules (anabolic reaction). The energy needed to drive the synthetic reactions comes from the breakdown reaction and the enzymes, which may number about 1000 in a single cell, are involved in its transfer. Action of enzymes on their substrates is often used in the identification of bacteria; the enzyme urase, breaks down urea into ammonia and carbon dioxide. The ammonium carbonate so formed can easily be detected in the growth medium by virtue of its alkalinity. The most obvious effect of oxygen on the bacterial cells depends on whether it is used as the final hydrogen acceptor in its respiratory process i.e. aerobic respiration. Bacteria which can grow only in the absence of free oxygen are termed anaerobes and bacteria which can switch to alternative respiratory or energy yielding pathways and therefore grow with or without oxygen are termed facultative anaerobes. Yet, another group of bacteria grow best at reduced oxygen levels and these are called microaerophillic. All bacteria seems to need some carbon dioxide in the atmosphere and most grow more readily when a relatively high concentration (5-10%) of carbon dioxide is supplied irrespective of their requirements for oxygen. 

 

Sterilisation

There are three classes: 

The physical method

  • Radiation: the commonest form of electromagnetic radiation used in microbiology are UV light and the much more energetic gamma rays. Exposure to direct sunlight slowly kills bacteria and is due to ultraviolet rays which occur at the extreme limits of the visible spectrum (350 - 750 nm).
  • Dry heat: dry heat at high temperature causes destruction of living cells and tissues by oxidation of their components. The extreme form is simply the incineration of flammable and disposable articles and their contaminating microorganisms e.g. carcasses of infected animals. The most common application of dry moderate temperature is the use of hot air oven. This is used for materials that are unaffected by temperature of 160 - 180 degrees centigrade for which autoclave is unsuitable. Examples are dry glass wares and un-wettable materials such as powder, oil and waxes.
  • Moist heat (boiling water): a temperature of 100 degree centigrade will kill all non-sporing organisms within 10 minutes. Most spores will be killed in 30 minutes at this temperature. However, some spores resist boiling for several hours, but addition of 2% sodium carbonate increases the bactericidal effects of boiling water. Suitable for infected instruments if they are to be used immediately.
  • Steaming at 100 degree centigrade: this is used mainly to sterilise certain complex media where the constituents might be broken down (hydrolyzed) at temperatures above 100 degrees centigrade e.g. sugars (these are prepared in the laboratory and range from sucrose, to galactose, and to maltose which are used to inoculate microorganisms) and gelatins.
  • Steaming under pressure: e.g. use of autoclave.

 

The chemical method

Many chemical agents are referred to as disinfectants, a term that is applied to substances which destroy microorganisms on inanimate objects. Other terms with a similar meaning are germicides and bactericides. A disinfectant which is not injurious to human tissue is called an anti-septic; and chemicals which are used to prevent organisms growing in a sterile medium but do not kill them are called bacteriostats.

Chemical agents function as sterilising agents by the following lethal mechanisms:

  • Interfering with the enzymatic system of the organism (enzyme poisoning).
  • Disruption of the cell membrane of the bacteria cell.
  • Coagulation of proteins.
  •  

Examples of disinfectants are alcohol (100% is not used for sterilisation, 70% is used. This is achieved by adding 30ml of distilled water to 70ml of alcohol (Rc x Rv) / Oc. Where, Rc = required concentration, Rv = required volume, Oc = original concentration). Example of common disinfectants are alcohol, chloroform, chlorine, glycerol, phenol and cresols; quartenary ammonium compounds e.g. centrimide. 

 

The filtration method.  

 

Incubator

An incubator is a special chamber designed for growing microorganisms in the laboratory. It has compartments where culture media are placed and a thermometer inlet to measure temperature of the chamber. It also has a knob to vary temperature to desired range. Culture media are placed in the chamber after inoculation with microorganisms and the temperature knob is set at 37 degrees centigrade for most human pathogens for a period of 24 hours. 

Urethral swab, urine, high vaginal smear, otitis media, wind swab, faeces/ stool, CSF, pus, nasal swab and throat swab. All these can be cultured microscopically. 

 

Culture media

Nutrient agar are used for the cultivation of many easily grown organisms e.g. staphylococci and Escherechia coli. 

Preparation

  1. Melt the nutrient agar by steaming.
  2. Cool to 50 degrees centigrade and pour 15 - 20 ml aseptically into clean, sterile petri dishes.

Nutrient agar is the base medium for preparing blood and chocolate agar. 

 

Blood agar

It is used for the cultivation and differentiation of more delicate organisms e.g. streptococci and gonococci.  Method

  • Melt nutrient agar by steaming.
  • Cool to 50 degrees centigrade and add 5 - 10 parts sterile, defibrinated or oxalated horse/ human blood.
  • Pour 15 - 20 ml volume aseptically into Petri dishes.

 

MacConkey agar

It is used for differentiating intestinal organisms  into lactose and non-lactose fermenting organisms. The peptone constitutes the nutrient base solidified by the agar. Sodium taurocholate (bile salt) inhibits many gram positive organisms and lactose and the indicator (neutral red) differentiates the lactose  fermenting from the non-lactose fermenting micobe (bacteria).

 

Culture media can generally be classified into:

A. Universal media  

  1. Nutrient agar: it is a universal medium that is non fastidious. Fastidious organisms cannot grow in it. It is the medium of choice for antibiotic susceptibility testing.

 

 

Nutrient agar without microbes.

 

  1. Blood agar: you cannot see through it when it is properly prepared (this is how you differentiate it from MacConkey agar in a practical exam or oral exam). It is also a universal medium because it allows most bacteria to grow in it. However, it is a differential medium. It does not contain any indicator per se. What is found in this medium is the product of the addition of blood to agarose. Sheep red blood is the blood of choice; using human blood is wrong, but some folks use expired human blood from blood banks. The blood is added to agarose between 40-45 degrees centigrade. The blood must form 5 - 10 percent of the total volume of the culture media. Blood is very rich so it allows microorganisms to grow very well. This medium is an enriched medium which enhances microbial growth; however, a liquid (enriched) medium is called an enrichment medium. Blood agar is a differential medium because some micro organisms have ability for hemolysis by secreting hemolysin (alpha and beta) which hemolyses red blood cells. Bacteria incapable of producing hemolysin do not hemolyse red blood cells. Non-hemolysing bacteria are designated to have gamma hemolysin because they cause gamma hemolysis (this produces a brownish appearance of the blood medium). Beta hemolysin producing bacteria will produce a translucent area around their colony in the blood medium. Alpha producing hemolysin will have a greenish tint in the blood medium; alpha hemolysin is a partial hemolyser. 
  2. A.            B.  
  3. Blood agar showing a cultured bacteria colony.
  4. Blood agar without bacteria in it.

 

B. Selective media  

  1. MacConkey agar: you can can see through this; it is translucent (differentiating factor from blood agar). It contains two important components called the agarose (it is the solidifier of all media) which dissolves at 60 degrees and solidifies at 45 degrees, and nutrients. It is selective for gram negative bacteria. Gram positive bacteria is not likely to grow here. It is also a differential medium meaning different organisms behave differently in it. It contains lactose. A lactose fermenting bacteria will ferment lactose to lactic acid. The non-lactose fermenting bacteria cannot ferment lactose. A lactose fermenter will come out as pink with a translucent environment in this media. Basis of operation is the conversion of lactose to lactic acid. The pink colour is due to an indicator (neutral red) which reveals the pH change in the medium.

 

  1.             B.  
  2. MacConkey agar showing colony of a lactose fermenting bacteria.
  3. MacConkey agar without the presence of bacteria.

 

  1. Cysteine lactose electrolyte deficient agar (CLED): this is used for culture in suspected causes of urinary tract infection (sample is urine). It is a selective and differential medium. It is used to differentiate between lactose and non-lactose fermenting bacteria. Indicator used it bromothymol blue. A lactose fermenting bacteria takes a yellowish appearance in this medium. 

 

CLED agar without microbes.

 

  1. Chocolate agar: it is the same thing as blood agar, but it is heated. It is introduced at 60 degrees centigrade into the agarose in order to lyse the red blood cells. Certain factors (X and V) are important for the culture of haemophilus. Alpha hemolysis is best seen on chocolate agar.

 

 

Chocolate agar without microbes.

 

Exams

In the practical exam, if you see chocolate agar, it is alpha hemolysis until proven otherwise (alpha hemolysin is responsible). If you see blood agar, it is beta hemolysis until proven otherwise (beta hemolysin is responsible). 

 

Examples of organisms responsible for alpha/ beta hemolysis?: This will be given in class.

 

Diseases that can be caused by bacteria? Will also be given in class

 

Antibiotics of choice? Come to his class.   

Microscope

Identify: binocular compound light microscope.

Principle: magnification.

Use: for magnification of specimens on a slide. 

Precautions

  • Clean the objective lens with xylene after each use.
  • Put off the light after use.
  • Cover or put the microscope in a box after use.

Terminologies

  1. High power field (magnification above x40)
  2. Low power field (magnification below x40).
  3. Oil Immersion lens (x100). This is immersed in oil before use.

 

Bijou bottle: it is used for specimen collection and biochemical tests. Bijou bottles are also used to prepare transport media to maintain the integrity of the sample taken. 

 

Wire loop: for inoculation.

 

Hand gloves (Identify/ name and use).

 

Universal bottle: this is used for a wide range of sample collection including saliva, urine, stool, etc.