L number Lab 2 Done by حسام أبو عوض Corrected by Mahdi sharawi Doctor
In many cases we need to identify the type of bacteria causing an infection in order to be able to choose the right medication (antibiotic). For this identification to be possible we need to obtain a pure isolated sample of our bacteria and this is what we will be discussing here. Media If I leave my sample (e.g. urine) at room temperature (and normal conditions) all the bacteria will die in 1 hour or even 30 minutes, so I need to supply the bacteria with a nutritional medium in which they can survive. Media is the substance in which some organisms (bacteria) are grown. So, we have to make a mixture that supplies our bacteria with all their requirements. The most common components of a media are: sugar, nitrogen, peptone and distilled water (there are many others, but these are the main ones). The media we prepare can be liquid (placed in test tubes) or solid (placed in petri dishes). Each type of media has its own uses. The liquid media is also called broth. When the broth is clear this means that there is no bacterial growth or contamination in our media. Turbidity (معكر) in the liquid means that there is bacterial growth, if this growth is not intentional then our media has been contaminated and we need to get rid of it. In solid media, bacterial growth is seen as appearance of spots, these are bacterial colonies. Solid media is made by adding agar (a gel-like substance which freezes at room temperature) to our mixture. Companies manufacture media; we only get them bottled and firmly secured (media would be in a powder form). On the bottle all the required information are written, this includes the name of the bacteria that can grow in this media, how to prepare the media, how to store it, etc. General preparation steps - Add the amount of media as instructed on the bottle [e.g. 50.2 grams for each 1000 ml of distilled water (try to be accurate)]. Never use tap water for it contains salts and many elements that would alter the nutrient content of the media and might damage the bacteria. - Put the media on an automatic heater with a magnetic stirrer. Usually this step takes around 30 minutes till we get a completely homogenous
solution with no conglomerates (تكتالت) of the media powder. Close the flask firmly. - Place an autoclave tape around your flask (or whichever bottle containing your media) then place your flask in the autoclave machine The required conditions in the autoclave machine.(اشي زي طنجرة الضغط) are a temperature of 121ᵒC and a pressure of 15 pounds/inch. Usually we keep our flask in the autoclave machine for around 30 minutes, but this differs according to the media so follow the instructions on the bottle. - When the autoclaving cycle is over take your flask out and look at the autoclave tape, if black bands appeared this means that the required temperature and pressure were reached and that our job was done perfectly [if not throw your media away and start all over again]. - Allow the media to cool down to 50-60ᵒC, this usually takes around 20-30 minutes but can vary according to the room temperature so keep monitoring the temperature using a thermometer (if you pour hot media in a plastic petri dish the dish will melt). - Before pouring our media in the petri dishes (or test tubes) we need to clean our working area, use antiseptics and turn the Bunsen burner on. - Now pour your media. If you are pouring it in a petri dish only fill about two-thirds of the dish. (Not full, to allow air to enter. Most bacteria are aerobic and need oxygen. Also, not less than covering the entire lower surface, because some of the media might evaporate so if we put too little we will have no media left). - To make a blood agar plate, before pouring the media and after cooling it, we add 5% sheep blood to the agar media (human blood can be used but sheep s is the best for bacterial growth). [if we pour blood on hot agar red blood cells break down and we lose them]. - To make chocolate agar we add the blood to the hot agar (once it is removed from the autoclave machine) so lysing the red blood cells and releasing their contents to the media. Some bacteria (e.g. Haemophillus influenzae and Nisseria) only grow in such a media where X-factor and V- factor molecules (released from red blood cells) are available for them. This media is called chocolate agar because its colour turns brownish.
Types of Media 1- Enriched Media: This media has many contents enhancing bacterial growth. Most common are blood agar and Mueller-Hinton media, around 99% of all bacteria can grow in these two media. Mueller-Hinton media is more sensitive than blood agar. 2- Selective Media: This media selects only one group of bacteria (not one species) or bacteria with a common feature between them. [E.g. enterobacteria are bacteria that live inside our bodies and can be identified in the MacConkey agar. The S.S. agar (salmonella-shigella agar) only detects salmonella and shigella]. 3- Differential Media: This media differentiates between the different types of bacteria within the same group using indicators (added to the media). {following are important examples} - S.S. Agar: an indicator is added and the salmonella appears in a yellow background with black dots while shigella appears with colourless dots (Salmonella reduces H₂S while shigella does not). - MacConkey Agar: Some enterobacteria can ferment lactose while others cannot. We add an indicator and we get two colours, a pinkish colour, these are the lactose fermenter bacteria, and a yellow/colourless colour, these are the non-lactose fermenter bacteria. - Mannitol Salt Agar: This agar is sensitive to staphylococcus bacteria only, but there are many species of this bacteria so we add an indicator and get two groups: Staphylococcus aureus, seen in a yellow colour, and a group for all other staphylococcus species seen as colourless-white spots. Streak Plate Method This is the method we use to get pure samples of bacterial colonies. - We receive a sample and we would have our petri dish ready for bacteria to be grown on it. - Take a wire loop (also known as nichrome wire) and pass it over the flame of a Bunsen burner to sterilize it.
- Now let the wire loop cool. After it has cooled down dip it in the sample to obtain just one drop of it. - On the upper quarter of your petri dish spread the sample in a zig-zag like pattern. - Now pass your wire loop over the flame again to sterilize and kill any bacteria remaining on the wire. - Rotate the petri dish 90ᵒ and from the end of the previous zigzag pattern drag (gently) and make another similar pattern. Repeat the previous two steps till there is just some little space left in the center of the petri dish (this is to allow bacteria to spread). - Now cover your petri dish with its cover and incubate it at the required temperature and for the needed time duration. After the incubation you get the following pattern: As you can see, the latter repeats of our zig-zag patterns always have less number of bacteria than former ones and this continues till we can see single colonies in the final region.