Aiding in the determination of the type of microorganism, microbiologists use different procedures to help them identify it.

They use gram stain, then special stains like ( Acid-fast staining, flagella stain) they check the morphology of the colony, and at last they do further biochemical tests to prove the result. acid-fast staining is used with Mycobacterium species. Because such species have dense lipid wall (waxes) that prevents the stains from penetrating the cell so we have to force the stain in. In this procedure, Carbol fuchsin is first driven into the bacterial cell using heat. The heat softens the waxes, enabling the stain to penetrate. Motility if a bacterium is able to swim its said to be motile. Bacteria unable to swim are said to be non-motile. Its often determined by the presence of flagella or axial filaments, but bacteria never possess cilia. A flagella stain can be used to demonstrate the presence, number, and location of flagella on bacterial cells. Motility can be demonstrated by one of two ways: 1- Stabbing the bacteria into a tube of semisolid agar. 2- Using the hanging-drop technique. 1- Incubation of bacteria in a tube of semisolid agar
Growth of bacteria in agar produces turbidity (cloudiness). Non-motile organisms will grow only along the stab line (thus, turbidity will only be seen along the line. Motile organisms will spread away from the line producing turbidity throughout the medium. (Check picture)
- Burtons microbiology for the health sciences- 9th edition. Page61

Solid agar poured on round plates (seen in lab) are used to cultivate bacteria, and then semisolid media (agar) is used to check the motility of microorganisms. By using the lab tools (e.g. Loop, needle) 1- Incubate the bacteria in the tube containing semisolid agar vertically. 2- Pull out

the loop from the tube in the same line it was insert by. 3- Wait about 24 hours so the growth pattern is determined. 4- If there is diffusion in the growth line after incubation this indicates that the microorganism is motile (because there are spaces between the agar particles which allow for movement), if the growth line is straight this indicates non-motile microorganism. In liquid media only growth of microorganism can be seen (cloudy media), but one cannot differentiate between motile and non-motile microorganisms. 2- Using the hanging-drop technique

Hanging-drop technique uses slides with depression covered with slip. This technique does not kill the microorganism like the gram stains, but instead it keeps it alive allowing the bacteria to swim in media. 1- Put a drop of liquid containing microorganisms on the coverslip. 2- Place the slide on the coverslip with the depression over the drop. 3- Flip the slide upside down (the drop should look like its hanging on the coverslip). Determination of motility is noticed by the direction of bacterial movement in the liquid. if the bacterial movement is with the liquid particles It indicates non-motile microorganism. If random movement is seen it indicates motile microorganism (e.g. Proteus, it has flagella all over its body thus results in random movements). Colony morphology Morphology of the bacterial colony that can be seen on the agar solid plate determines the type of microorganism. The clusters of cells on the plate are called colonies that present millions of microorganisms resulting from the cultivation of a single cell. Two tests besides checking the morphology of the colony are used to determine the type of colony,

the gram stain test and biochemical tests. The morphology of a Bacterial colony are of many aspects (e.g. shape, color, texture shiny or mate-). By determining these aspects, experienced specialist can distinguish different type of colonies or different type of microorganisms. Also enzymatic activity can be noticed on various types of media, enzymes may possess catalase activity, coagulate activity or cytochrome oxidative activity. These activates can be noticed upon the use of indicators, if positive, change in color or formation of bubbles will occur pointing for different types of microorganisms. (These enzymes will be further discussed in details later) biochemical tests are done after that to finalize the result. Size of colonies is determined by the organisms rate of growth (generation time), It is the time for the mother cell to divide into two daughter cells. Time differs from one type of microorganism to another. In mycobacterium it takes 16 hours to divide while in E-coli it takes only 15 minutes.

Atmospheric Requirement In the microbiology laboratory, it is useful to classify bacteria on the basis of their relationship to oxygen and carbon dioxide. With respect to oxygen, a bacterial isolate can be classified into one of five major groups: obligate aerobes, microaerophilic aerobes, facultative anaerobes, aerotolerant anaerobes, and obligate anaerobes (check picture below)

1- Obligate aerobes require oxygen, it require an atmosphere containing 20% to 21% oxygen, because it uses aerobic respiration and the electron transport chain needs O2 as a final receptor to produce ATP. Obligate aerobes have the enzymes necessary to break down the free radicals (substances that cause aging in human body) resulting from the aerobic respiration thus they can survive in the presence of Oxygen. (e.g. Mycobacteria and certain fungi). 2- Microaerophiles require reduced oxygen concentrations ( usually around 5%) for multiplication. Nisseria gonorrboeae (the causative agent of gonorrhea) and campylobacter spp. (which are major causes of bacterial diarrhea) are examples of microaerophilic. 3- Facultative anaerobes, anaerobes can be defined as organisms that do not require oxygen for life and reproduction. However, they vary in their sensitivity to oxygen. Facultative anaerobes are capable of surviving in either the presence or absence of oxygen (anywhere from 0% O2 to 20% O2). (E.g. members of the family Enterobacteriaceae, most streptococci, most staphylococci). 4- Aerotolerant Anaerobes does not require oxygen, grows better in the absence of oxygen, but can survive in atmospheres containing molecular oxygen (such as air and CO2 incubator) , the concentration of oxygen that an aerotolerant anaerobes can tolerate varies from one species to another. 5- Obligate anaerobes is an anaerobe that can grow in an anaerobic environment, it doesnt have the enzymes required for the breakage of free radicals resulting from the aerobic respiration thus it cant survive atmospheric containing oxygen. - In general, obligate anaerobes, aerotolerant anaerobes, and facultative anaerobes can thrive in an atmosphere devoid of oxygen. - Anaerobic respirators use anything but O2 as a final receptor in the electron transport chain. - Capnophiles grow better in laboratory in the presence of increased concentrations of CO2 (5% 10%) e.g., bacteroides and Fusobacterium species (anaerobes) , e.g., certain Nisseria, Campylobacter, and Heamophilus species (aerobes)

Nutritional requirements All bacteria need some form of the elements carbon, hydrogen, oxygen, sulfur, phosphorus and nitrogen for growth. Some species demand special elements such as potassium, calcium, iron, zinc. Certain microorganisms have specific vitamin requirement and some need organic substances secreted by other living microorganisms during their growth. Organisms with especially demanding nutritional requirements are said to be fastidious; think of them as being fussy. The nutritional needs of a particular organism are usually characteristic for that species of bacteria and sometimes serve as important clues when attempting to identify the organism. E.g. Heamophilus influenzae need special factors in order to grow. Bacteria causing laryngitis loves blood, it destroys RBCs to aid on the nutrients inside. So its cultivated on a blood plate (basic medium + 5% blood), sometimes its boiled to let the RBCs replicate and increase in number resulting in something called Chocolate agar (brown in color). It took microbiologists long time in research of the nutritional needs for every type of bacteria or microorganism. For instance the legionella (bacteria causing pneumonia) was discovered in a conference in Philadelphia when around 35 people of 300 caught strange symptoms and died of it. Microbiologists took samples from all over the place and discovered that the legionella lived on the coal (dust) particles stuck on the Air conditioners filters. From that they discovered that legionella demand coal in its environment. Biochemical and metabolic activities As bacteria grow, they produce many waste products and secretions, some of which are enzymes that enable them to evade the host and cause the disease. Microorganisms secrete different type of enzymes that reveal its type. To aid in the identification of certain types of bacteria in the lab, they are inoculated into various substrates (e.g., carbohydrates and amino acids) to determine whether they possess the enzymes necessary to break down those substrates or not. For example aerobic microorganisms have catalase enzymes that break down the free radicals resulting from aerobic respiration. Staphylococci produce coagulate enzyme.

Some bacteria are characterized by the production of certain gases, such as carbon dioxide, hydrogen sulfide, oxygen, or methane. The H2F gas when its produced turn the media black, CO2 when produced, it pushes the agar up creating a gap at the bottom of the tube. Change in color when testing for lactose or glucose fermentation determines the result whether its positive or negative and thus leading to identification of the type of bacteria. We will learn in laboratory how to look for these enzymes in order to define the type of bacteria.

Pathogenicity Bacteria exists everywhere, we drink bacteria, inhale bacteria, ingest bacteria. The vast majority of these bacteria are not harmful, and little are pathogenic. Many pathogens are able to cause disease because they possess capsules, pili, endotoxins (biochemical components of the cell walls of Gram-negative bacteria), exotoxins or exoenzymes that damage cells and tissues. There are two kinds of pili: 1- attachment pili (fimbriae) 2- Long conjugation pili, also called "F" or sex pili. The attachment pili attach the bacteria on the host cell, usually short and quite numerous and enable bacteria to colonize environmental surfaces or cells and resist flushing. The sex pili that are longer and very few in number, enables conjugation. Conjugation is the transfer of DNA from one bacterium to another by cell-to-cell contact. In gram-negative bacteria it is typically the transfer of DNA from a donor or male bacterium with a sex pilus to a recipient or female bacterium to enable genetic recombination. Another characteristic that helps the pathogen is the capsule. The capsule provides the bacteria with camouflage from the antigens on the surface of the host cell wall, also from the phagocytic cells

(macrophages, neutrophils). Neutrophils are more abundant in number but less effective as a phagocytic. Macrophages are less in number but far more effective. When the bacteria replicate and become large in number it possess two kind of toxins 1- endotoxins ( Gram-negative bacteria) 2- exotoxins ( gram-positive bacteria) In some cases you might find the two types of toxins in one bacteria but its rare. The endotoxins are part of the outer membrane of the gramnegative bacteria( lipid A toxin- embedded in the saccharide membrane). When bacteria replicate it realases lipid A which is an endotoxin. E-coli can produce endotoxins in the bloodstream causing toxaemia. Exoenzymes are another harmful characteristic possessed by the bacteria. E.g. collagenase, and hydrogenase . the matrix of the cell consist of two important components: collagen and hydronic acid, these two components aid in giving the youthful look on the skin and collagen forms the joint. Exoenzymes like collagenase and hydrogenase are secreted by pathogens like staphylococcus aureus resulting in breakage of the cell matrix. Bacteria invade the cell through the space, replicate and spread all over. All these characteristics used by the bacteria are called virulent factors that aid in destroying the cell. Frequently, pathogenicity is tested by injecting the organism into mice or cell cultures. Table 4-6 in book lists the common pathogenic bacteria (dont know if we should memorize the table but better if you take a look at it).

Genetic composition Most modern labs are moving toward the identification of bacteria using some type of test procedure that analyzes the organisms DNA or RNA. These tests are called molecular diagnostic procedures. Its a much faster technique and needs no biochemical or staining tests. Its very common in the epidemic cases. Steps : 1- Isolate a DNA probe (single stranded DNA sequence) of a microorganism. 2- Compare it with known bacterial sequences 3- Identify the bacteria Also, through the use of 16s rRNA sequencing, a researcher can determine the degree of relatedness between two different bacteria. Unique bacteria Some kind of bacteria doesnt have the same attributes possessed by the typical bacteria, thus they are often reffered to as unique or rudimentary bacteria. This kind of bacteria lives only inside the cell and because they are so small and difficult to isolate, they were formely thought to be viruses.

Special thanks : By mohammad malik " You better find a Malaysian friend to translate for you XD " Terima kasih banyak2 Baraa Srouji sebab tolong aku buat lecture note ni.Aku dapat belanja kau teh dengan nescafe jela. Bashar fares Terimakasih untuk pandu uji =) Yazan Ashhab Terbaik ahh drift kau :D Abu Al lool abada Kau lah kawan dunia akhirat aku. Ukhuwah fillah abadan

Abdulrhman al dorii AKA DJ_DORI: kalau lah wujud dua orang macam kau dalam dunia ni, kitorang akan ..XD Aya Rihan Kau sorang yang optimistic so aku senang berkawan ngn kau Zeina hammad ( C-town ) Terima kasih kerana meghidupkan group ni, kekal aktif dan teruskan lawak2 bodoh kau sebab kadang2 kelakar gak Abu AL Louzi (Balduna) Aku bangga ah ngan kau even kau nerd pun. Cool kot =) Huzaifa Talbani Terima kasih Cd kau. Kitorang tak gune pun. Thanks gak untuk dinner tuh :D Nadhirah Nadora our " Google Translator amazing friend :)