Micro Lab Manual for Midterms Part 2 PDF

**PART** 1 **LEARNING OBJECTIVES** *Once you* have completed the experiments *in* this section*,* you *should* be *able to* **1.** Identify the laboratory equipment and culture media needed to develop and maintain pure cultures. **2.** Identify the types of microbial flora that live on the skin...

**PART** 1 **LEARNING OBJECTIVES** *Once you* have completed the experiments *in* this section*,* you *should* be *able to* **1.** Identify the laboratory equipment and culture media needed to develop and maintain pure cultures. **2.** Identify the types of microbial flora that live on the skin and explain how hand washing affects them. successful subculturing of microorganisms. **4.** Explain streak-plate and spread-plate isolation of microorganisms from a mixed microbial population for subsequent pure culture isolation. 5\. Identify cultural and morphological characteristics of microorganisms grown in pure culture. Microorganisms are ubiquitous. We find them in soil, air, water, food, and sewage, and on body surfaces. In short, every area of our environment is replete with them. Microbiologists separate these mixed populations into individual species for study. A culture containing a single, unadulter- ated species of cells is called a **pure culture**. To isolate and study microorganisms in pure culture, microbiologists require basic laboratory equip- ment and apply specific techniques, as illustrated in **Figure P1.1**. **Media** The survival and continued growth of microorgan- isms depend on an adequate supply of nutrients and a favorable growth environment. For survival, most microbes must use soluble, low-molecular- weight substances that are frequently derived from the enzymatic degradation of complex nutrients. A solution containing these nutrients is a **culture medium**. All culture media are liq- uid, semisolid, or solid. A liquid medium lacks a solidifying agent and is called a **broth medium**. A broth medium is useful for cultivating high numbers of bacterial cells in a small volume of medium, which is particularly helpful when an assay requires a high number of healthy bacte- rial cells. A broth medium supplemented with a solidifying agent called **agar** results in a solid or semisolid medium. Agar, an extract of seaweed, is a complex carbohydrate composed mainly of galactose, and is without nutritional value. Agar serves as an excellent solidifying agent because it liquefies at 100°C and solidifies at 40°C. Because of these properties, we can cultivate organisms, especially pathogens, at temperatures of 37.5°C or slightly higher without fear of the medium liquefy- ing. A completely solid medium requires an agar concentration of 1.5% to 1.8%. A concentration of less than 1% agar results in a **semisolid medium**. A semisolid medium is useful for testing a cell\'s ability to grow within the agar at lower oxygen **1** Broth Semisolid Agar slant Agar deep **Equipment** Media Autoclave Bunsen burner Microincinerator Culture tubes Petri dishes Wire loops and needles Pipettes **Pure culture techniques** Incubators Refrigerators Streak plate } Cultivation chambers Isolation of pure cultures **Figure P1.1 Laboratory apparatus and culture techniques** levels and for testing the species\' motility. A solid medium is advantageous because it presents a hardened surface on which microorganisms can be grown using specialized techniques for the isolation of discrete colonies. Each **colony** is a cluster of cells that originates from the multiplica- tion of a single cell and represents the growth of a single species of microorganism. Such a defined and well-isolated colony is a pure culture. Also, while in the liquefied state, we can place solid media in test tubes, which then cool and harden in a slanted position, producing **agar slants**. These are useful for maintaining pure cultures. The slanted surface of the agar maximizes the available surface area for microorganism growth while minimizing the amount of medium required. Similar tubes that, following preparation, harden in the upright position are designated as **agar deep tubes**. Agar deep tubes are used primarily for studying gaseous requirements of microorgan- isms, since gas exchange between the agar at the butt of the test tube and the external environment is impeded by the height of the agar. Liquid agar medium can also be poured into Petri dishes, pro- ducing **agar plates**, which provide large surface areas for the isolation and study of microorgan- isms. The various forms of solid media are illus- trated in **Figure P1.2**. Side view Front view **(a)** Agar slants **Figure P1.2 Forms of solid (agar) media** **2** **Part 1** L **(b)** Agar deep tube **(c)** Agar plate **Heat** Moist (wet heat) **Filtration** Cellulose-acetate membrane filters with pore sizes in the range of 8.0 μm to less than 0.05 μm Ethylene oxide **Chemicals** **Radiation** lonizing **Figure P1.3 Sterilization techniques** empty glassware, glass pipettes, and glass syringes Free-flowing steam at 100°C (intermittent sterilization); for thermolabile solutions (e.g., Plastic dishes and pipettes Living tissues Plastic pipettes and Petri dishes In addition to nutritional needs, we must regu- late environmental factors, including proper pH, temperature, gaseous requirements, and osmotic pressure. You can read a more detailed explana- tion about the cultivation of microorganisms in Part 4; for now, you should simply note that numerous types of media are available. **Aseptic Technique** Sterility is the hallmark of successful work in the microbiology laboratory, and **sterilization** is the process of rendering a medium **or** material free of all forms of life. To achieve sterility, it is mandatory that you use sterile equipment and employ **asep- tic techniques** when handling bacterial cultures. Using correct aseptic techniques minimizes the likelihood that bacterial cultures will be contami- nated, and reduces the opportunity that you will be exposed to potential pathogens. **Figure P1.3** is a brief outline of the routine techniques used in the microbiology laboratory, and you will learn more about the control of microorganisms in Part 9. **Culture Tubes and Petri Dishes** We use glass **test tubes** and glass or plastic **Petri dishes** to cultivate microorganisms. We can add a suitable nutrient medium in the form of broth **or** agar to the tubes, while we use only a solid medium in Petri dishes. We maintain a sterile environment in culture tubes by various types of closures. Histori- cally, the first type, a cotton plug, was developed by Heinrich G. F Schröeder and Theodor von Dusch in the nineteenth century. Today most laboratories use sleeve-like caps (Morton closures) made of metal, such as stainless steel, or heat-resistant plastics. The advantage of these closures over the cotton plug is that they are labor-saving and, most of all, that they slip on and off the test tubes easily. Petri dishes provide a larger surface area for growth and cultivation. They consist of a bottom dish portion that contains the medium and a larger top portion that serves as a loose cover. Petri dishes are manufactured in various sizes to meet different experimental requirements. For routine purposes, we use dishes approximately 15 cm in diameter. The sterile agar medium is dispensed to previously sterilized dishes from molten agar deep tubes containing 15 ml to 20 ml of medium, or from a molten sterile medium prepared in bulk and contained in 250-, 500-, and 1000-ml flasks, depending on the volume of medium required. When cooled to 40°C, the medium will solidify. Remember that *after inoculation, Petri dishes are incubated in an inverted position* (top down) to prevent condensation formed on the cover dur- ing solidification from dropping down onto the surface of the hardened agar. For this reason, we should label Petri dishes on the bottom of the dish. This makes it easier to read the label and minimizes confusion if two Petri dish covers are interchanged. **Figure P1.4** illustrates some of the culture vessels used in the laboratory. Built-in ridges on tube closures and Petri dishes provide small gaps necessary for the exchange of air. Microorganisms must be transferred from one ves- sel to another, **or** from stock cultures to various media, for maintenance and study. This transfer **Part 1** A B C D E A. Bacteriological tube B. Screw cap C. Plastic closure **(a)** Test tube rack with tubes showing various closures **Figure P1.4 Culture vessels** **(b)** Petri dish **(c)** DeLong shaker flask with closure is called **subculturing**, and must be carried out under aseptic conditions to prevent possible contamination. **Wire loops** and needles are made from inert metals such as Nichrome or platinum and are inserted into metal shafts that serve as handles. They are extremely durable instruments and are easily sterilized by incineration in the blue (hot- test) portion of the Bunsen burner flame. A wire loop is useful for transferring a small volume of bacteria onto the surface of an agar plate or slant. We use a needle to inoculate a culture into a broth medium or into an agar deep tube. A **pipette** is another instrument used for aseptic transfers. Pipettes are similar in function to straws; that is, they draw up liquids. They are glass or plastic, and drawn out to a tip at one end, with a mouthpiece forming the other end. They are calibrated to deliver different volumes depending on requirements. Pipettes may be sterilized in bulk inside canisters**,** or they may be wrapped individu- ally in brown paper and sterilized in an autoclave or dry-heat oven. A micropipette (commonly called a \"pipetter\") with a disposable, single-use plastic tip is useful for transferring small volumes of liquid (less than 1 ml). A **Cultivation Chambers** Part 4 discusses specific temperature require- ments for growth; however, a prime requirement for the cultivation of microorganisms is that they be grown at their optimum temperature. We use an **incubator** to maintain optimum temperature during the necessary growth period. It resembles an oven, and is thermostatically controlled so **4** **Part 1** Needle Shaft **(a)** Transfer needle **(e)** Micropipette **Figure P1.5 Transfer instruments** Etched ring Final few drops must be blown out to deliver indicated volume ابیلیسا لسلسلسلة سلسلة سلسال BAMBAM TD 1 IN 1/100 ml Identification and graduations 0.1 ml: major division 0.01 ml each: minor divisions 10 IN 1/10 ml TD 20° C 10 ml **(c)** Blow-out pipette pipette **Mechanical Pipette Aspirators** (**E)** No etched ring on mouthpiece (to deliver) **Part 1** **5** that temperature can be varied depending on the requirements of specific microorganisms**.** Most incubators use dry heat. Moisture is supplied by placing a beaker of water in the incubator during the growth period. A moist environment retards dehydration of the medium and thereby helps avoids misleading experimental results. A thermostatically controlled **shaking waterbath** is another piece of apparatus used to cultivate microorganisms. Its advantage is that it provides a rapid and uniform transfer of heat to the culture vessel, and its agitation provides increased aeration, resulting in acceleration of growth. The primary disadvantage of this instru- ment is that it can be used only for cultivation of organisms in a broth medium. Many laboratories also use shaking incuba- tors that utilize dry air incubation to promote aeration of the broth medium. This method has a distinct advantage over a shaking waterbath, since there is no chance of cross contamination from microorganisms that might grow in the waterbath. **Refrigerator** We use a refrigerator for a wide variety of pur- poses, such as maintaining and storing stock cultures between subculturing periods, and storing sterile media to prevent dehydration. It is also used as a repository for thermolabile solutions, antibiotics, serums, and biochemical reagents. **FURTHER READING** Refer to the section on microbial growth in your textbook for more information on materials and techniques utilized in the cultivation of bacteria. Search the index for the specific terms \"Agar,\" \"Colony,\" and \"Sterile.\" **CASE STUDY** **HAND WASHING AND ASEPTIC TECHNIQUE** A local microbiological testing laboratory service, Aureus Systems, notified its regional headquarters about a possible contamination issue in either its Quality Assurance/Quality Control (QA/QC) lab or in its testing center proper. As an outside adviser, you have been hired to investigate the situation and to monitor the laboratory procedures of this local branch. Upon your arrival, a senior lab tech- nician (John Doe) allows you to shadow him and answers your questions for the week of your visit. During your week, you notice some instances of gross indifference to standard laboratory practices concerning personal hygiene and personal protec- tion practices. On numerous instances you have recorded **Mr.** Doe removing his latex gloves and continuing to handle specimens and laboratory media without washing his hands. Many times, Mr. Doe has been reprimanded for this practice, as well as for fail- ure to wash his hands before leaving the lab room itself. **Mr.** Doe argues that his aseptic technique practices are at a high enough standard that he is incapable of contaminating any specimens that he is working on in the lab. On numerous occasions his supervisors have recorded that stock media preparations used by Mr. Doe and other labora- tory technicians have been contaminated with unknown microbes. The regional headquarters requires labora- tory proof that Mr. Doe---and not the equipment **or** the lab environment-is the source of the contamination. **Questions to Consider**: 1\. Why is it important to wash your hands BEFORE and AFTER using bacterial cultures? 2. How would you isolate the contaminating microbes from the contaminated stocks to determine what species they are? **3.** Why would the use of \"aseptic technique\" be important in a testing lab, or any microbiology lab? **6** **Part 1** **Effectiveness of Hand Washing** **EXPERIMENT** **LEARNING OBJECTIVES** *Once you have completed this experiment, you should* be *able to:* **1.** Differentiate between the residential flora and transient flora found on skin surfaces 2\. Determine the effect of hand washing on the reduction of organisms on the skin 3. Explain the effectiveness of using soap alone or soap accompanied by surgical brushing **Principle** Each day our hands come in contact with numer- ous objects and surfaces that are contaminated with microorganisms. These may include door handles, light switches, shopping carts, sinks, toi- let seats, books, or even things like compost piles or body fluids, to name a few. The lack of adequate hand washing is a major vehicle in the transmis- sion of microbial infection and disease. Our skin is sterile while *in utero* and first becomes colonized by a normal microbial flora at birth as it **is** passed through the birth canal. By the time you reach adulthood, your skin is calculated to contain 1012 (1,000,000,000,000), **or** one trillion, bacteria, most of which are found in the superficial layers of the epidermis and upper hair follicles. This normal flora of microorganisms is called the **resident flora**, the presence of which does not cause negative effects in healthy individuals. In fact, it forms a symbiotic relationship with your skin, which is vital to your health. This beneficial rela- tionship can change in patients who are immuno- compromised, or when residential flora accidentally gains entrance to the host via inoculating needles, indwelling catheters, lacerations, and the like. Microorganisms that are less permanent, present for only short periods, are termed **transient flora**. This latter flora can be removed with good hand washing techniques. Resident flora is more difficult to remove because it is found in the hair follicles and is covered by hair, oil, and dead skin cells that obstruct its removal by simple hand washing with soap. Surgical scrubbing is the best means for removal of these organisms from the skin. Surgical hand washing was introduced into medical practice in the mid-nineteenth century by the Hungarian physician Ignaz Semmelweis while working at an obstetric hospital in Vienna. He observed that the incidence of puerperal fever (childbirth fever) was very high, with a death rate of about 20%. He further observed that medical students examining patients and assisting in deliv- eries came directly from cadaver (autopsy) labora- tories without stopping to wash their hands. Upon his insistence, medical students and all medical personnel were required to wash their hands in a chloride of lime (bleach) solution before and after all patient contact. The incidence of death from puerperal fever dropped drastically to around 1%. Semmelweis\'s effort led to the development of routine surgical scrubbing by surgeons, which has become essential practice for all surgical proce- dures in modern medicine. **FURTHER READING** Refer to the sections on hand washing and laboratory hygiene to review proper laboratory protocols and microbe handling safety. In your textbook\'s index, search under the terms \"Hygiene\" and \"Aseptic Technique.\" CLINICAL APPLICATION **Preventing Nosocomial Infections** Nosocomial (hospital-acquired) infections are mainly transmitted from the unwashed hands of healthcare providers. Transient and residential flora on healthcare providers\' skin can infect hospital patients whose immune systems are compromised. The cornerstone for the prevention of nosocomial infections is meticulous hand washing and scrub- bing by healthcare personnel. In the laboratory setting, your normal flora may contaminate patient samples and skew your results, leading to a mis- diagnosis. It **is** important for everyone in the lab to correctly wash their hands before and after han- dling biological materials. **7** **AT** THE **BENCH** **Materials** **Media** 4 nutrient agar plates per student pair **Equipment** Liquid antibacterial soap 8 sterile cotton swabs 2 test tubes of sterile saline Microincinerator Glass marking pencil Surgical hand brush Quebec colony counter Stopwatch **Procedure** Lab One 1\. One student becomes the washer and the other student the assistant. **The washer must not wash hands before coming to the lab**. **2.** The assistant uses the glass marking pencil to label the bottoms of the nutrient agar plates. The assistant marks two plates as \"Water\" and two plates as \"Soap,\" and draws a line down the middle of each plate to divide each plate in half. For the \"Water\" plates, label the halves as R1, R2, R3, and R4. For the \"Soap\" plates, label the halves as L1, L2, L3, and L4. See **Figure 1.1**. **3.** The assistant aseptically dips a sterile cotton swab into the first test tube of sterile saline. To do this, complete the following steps. **a.** First, light the Bunsen burner. **b.** Uncap the test tube; after removing the cap, keep the cap in your hand with the inner aspect of the cap pointed away from your palm. The cap must never be placed on the laboratory bench, because doing so would compromise the aseptic procedure. **c**. Flame the neck of the tube by briefly pass- ing it through the flame of the Bunsen burner. the swab in the tube, soaking it with saline. Avoid touching the sides of the tube with the swab. The assistant then rubs the moistened cotton swab on the pad of the washer\'s **right** thumb. **4.** The assistant then aseptically inoculates the half of the nutrient agar plate labeled R1 by streaking the far edge of the plate several times, then making a zigzag streak on only the half labeled R1. See **Figure 1.2**. *Caution: Do not gouge the surface of the agar plate*. Water **Figure 1.2 Plate inoculation**. R1 R2 L1 L2 L3 L4 Water **Figure 1.1 Plate labeling** **8** **Experiment 1** Water Soap Soap the nutrient agar plate labeled R2 in the same way that R1 was inoculated. thumb for 2 minutes and then 3 minutes, respec- tively. The assistant uses a new **dry** sterile cotton swab each time, and aseptically inoculates R3 and R4, respectively. See **Table 1.1**. 7\. The assistant and washer now move to the left hand. The assistant aseptically dips the sterile cotton swab into the second test tube of ster- ile saline (following the process from step 3), rubs the moistened cotton swab over the pad of the left thumb, and aseptically inoculates L1 as shown in Figure 1.2. so that the washer can wet the thumb and index finger of the left hand under warm run- ning water. The assistant applies one or two drops of liquid soap to the thumb and index finger and the washer washes for 1 minute by rubbing the thumb over the index finger. Rinse well. Shake off water from the hand but do not blot dry. The assistant turns off the tap. The assistant then uses a dry sterile cotton swab to obtain a sample from the washed thumb pad and inoculates L2. **TABLE 1.1** **Inoculation of Nutrient Agar Plates** **SOAP-LEFT** Wash with soap 1 soap but also scrubbing the thumb with a sur- gical brush, for 2 minutes and then 3 minutes, respectively. The washer holds the surgical brush and the assistant adds saline to the brush to dampen it, and then adds one or two drops of soap to the thumb and also to the brush. *Caution: Place the brush bristles-up* on *a dry paper towel between washings*. The assistant uses a new dry sterile cotton swab each time, and aseptically inoculates L3 and L4, respectively. Refer back to Table 1.1. **10.** Incubate all plates in an inverted position at 37°C for 24 to 48 hours. **Procedure** Lab Two **1. Macroscopically.** Visually observe the presence of growth on the surface of each agar plate in each section. Record **your** results in your Lab Report as 0 = no growth, 1+ = slight growth, 2+ = moderate growth*,* 3+= heavy growth, and 4+= maximum growth. **WATER-RIGHT** **THUMB** **THUMB** R1 No wash, damp L1 No wash, damp cotton swab cotton swab R2 L2 dry cotton swab minute, dry cotton swab R3 Wash 2 minutes, **L3** Soap and surgical dry cotton swab brush 2 minutes, dry cotton swab Percent reduction R4 Wash 3 minutes, **L4** Soap and surgical dry cotton swab brush 3 minutes, dry cotton swab **3322** **=** \[Colonies (section 1) -Colonies (section x)\] Colonies (section 1) **X** = sections 2, 3, 4 for each hand **Experiment 1** **9** *This page intentionally left blank* Name: Date: Section: **EXPERIMENT** 1 **Lab Report** **Observations and** Results **1.** Record the macroscopic observations in the chart below. **Growth** = **none,** **Section** (**Water-** **1** + = **slight**, 2 += **moderate,** **Section** (**Soap-** **Growth** **(0 = none**, **1**+= **slight, 2**+= **moderate**, Right **Thumb**) **3**+= **heavy**, **4** + **= = maximum**) **Left** **Time** **3**+= **heavy**, **(min)** **4**+= **maximum)** R1 0 L1 R2 L2 1 R3 R4 **2** 3 L3 L4 3 **Section** **Section** **(Water-** Right **(Min**) **Number of Colonies** **Percent** **Time** **Number of** **Percent** **Reduction** **Thumb)** **(Min**) **Colonies** **Reduction** R1 0 L1 0 R2 1 L2 1 R3 **2** L3 **2** R4 L4 3 **Review Questions** 1\. Compare the effectiveness of hand washing with water, with soap, and with soap and surgical scrubbing. **11** **3.** How does hand washing affect residential versus transient flora? 4\. Why do you think hand washing is necessary when medical and surgical personnel wear gloves dur- ing surgery and when examining patients? **12 Experiment 1: Lab Report** **Culture Transfer Techniques** **EXPERIMENT** 2 **LEARNING OBJECTIVES** *Once you have completed this* experiment*, you should be able to* 1\. Perform the technique for aseptic removal and transfer of microorganisms for subculturing. 2\. Correctly sterilize inoculating instruments in a microincinerator or the flame of a Bunsen burner. **3.** Correctly remove and replace the test **Principle** We transfer microorganisms from one medium to another **by subculturing.** This technique is used routinely in preparing and maintaining stock cultures, as well as in microbiological test procedures. Microorganisms are always present in the air and on laboratory surfaces, benches, and equipment. These ambient microorganisms can serve as a source of external contamination and interfere with experimental results unless proper aseptic techniques are used during subculturing. Described below are essential steps that you must follow for aseptic transfer of microorganisms. **Figure 2.1** illustrates the complete procedure. 1\. Label the tube you will inoculate with the name of the organism and your initials. will inoculate in the palm of your hand, secure with your thumb, and separate the two tubes to form a V in your hand. 3\. Sterilize an inoculating needle or loop by hold- ing it in the microincinerator or the hottest portion of the Bunsen burner flame until the wire becomes red hot. Once sterilized, hold the loop in your hand and allow it to cool for 10 to 20 seconds; never put it down. 4\. Uncap each tube by grasping the first cap with your little finger and the second cap with your next finger and lifting the closure upward. *Note: Once removed, these caps must be kept* *in the hand that holds the sterile inoculat- ing loop or needle; the inner aspects of the caps point away from the palm of the hand*. Never place the caps **on** the laboratory bench, because that would compromise the aseptic procedure. mouths of the tubes by briefly passing them through the opening of the microincinerator **or** through the Bunsen burner flame two to three times rapidly. Cool the sterile transfer instrument further by touching it to the sterile inside wall of the culture tube before removing a small sample of the inoculum. 6\. Depending on the culture medium, a loop or needle is used for removal of the inoculum. Loops are commonly used to obtain a sample from a broth culture. Either instrument can be used to obtain the inoculum from an agar slant culture by carefully touching the surface of the solid medium in an area exhibiting growth so as not to gouge the agar. A straight needle is always used when transferring microorgan- isms to an agar deep tube from both solid and liquid cultures. **b.** For a broth-to-slant transfer, obtain a loop- obtain the inoculum from the agar slant. Insert a straight needle to the bottom of the tube in a straight line and rapidly withdraw along the line of insertion. This is called a stab inoculation. 7\. Following inoculation, remove the instrument 8\. Replace the caps on the same tubes from which they were removed. remaining organisms. **13** **PROCEDURE** **1** Label the tube to be inoculated with the name of the organism and your initials. 2 Place the tubes in the palm of your hand, secure with your thumb, and separate to form a V. **3** Flame the needle or loop until the wire is red. **4** With the sterile loop or needle in hand, uncap the tubes. **5** Flame the necks of the tubes by rapidly passing them through the flame once. **6 Slant-to-broth transfer:** Obtain inoculum from slant and dislodge inoculum in the broth with a slight agitation. **Slant-to-agar deep transfer:** Obtain inoculum from slant. Insert the needle to the bottom of the tube and withdraw along the line of insertion. 7 Flame the necks of the tubes by rapidly passing them through the flame once. **Figure 2.1 Subculturing procedure** **14** **Experiment 2** **8** Recap the tubes. **9** Reflame the loop or needle. In this experiment, you will master the manip- ulations required for aseptic transfer of micro- organisms in broth-to-slant, slant-to-broth**,** and slant-to-agar deep tubes. You will use a positive and a negative control to test your ability to main- tain aseptic techniques while transferring cultures. Experiment 3 discusses the technique for transfer to and from agar plates. **FURTHER READING** Refer to the section on aseptic culture techniques in your textbook; more information on culturing technique practices in the microbiological labora- tory will be reviewed. In your textbook\'s index, search for the terms \"Aseptic Technique\" and \"Sterile.**\"** CLINICAL APPLICATION **Aseptic Inoculation and Transfer** It is mandatory that microbiology laboratory work- ers learn and perfect the skill of inoculating bacte- rial specimens on agar plates, in liquid broth, or in semisolid medium, and be able to subculture the organism from one medium to another. A sterile inoculating needle or loop is the basic instrument of transfer. Keep in mind that transferring bacterial cultures requires aseptic or sterile techniques at all times, especially if you are working with pathogens. Do not contaminate what you are working with and do not contaminate yourself. **AT** THE **BENCH** **Materials** **Cultures** Twenty-four-hour nutrient broth and nutrient agar slant cultures of *Serratia marcescens* and a sterile tube of nutrient broth. The nutri- ent broth tubes will be labeled \"A\" and \"B,\" and the contents will be known only by the instructor. **Media** Three nutrient broth tubes Three nutrient agar slants Three nutrient agar deep tubes **Equipment** **Procedure** Lab One 1\. Label all tubes of sterile media as described in the Laboratory Protocol section on page xv. 2. Following the procedure outlined and illus- trated previously (Figure 2.1), perform the following transfers. *nutrient agar deep tube, and nutrient broth.* **b.** *Broth culture \"B\" to a nutrient agar slant**,*** *nutrient agar deep tube, and nutrient broth.* *nutrient agar slant, nutrient agar deep tube, and nutrient broth**.*** **3.** Incubate all cultures at 25°C for *24* to 48 hours. **Procedure** Lab Two 1\. Examine all cultures for the appearance of growth, which is indicated by turbidity in the broth culture and the appearance of an orange-red growth on the surface of the slant and along the line of inoculation in the agar deep tube. vided in the Lab Report. 3\. Confirm your results with the instructor to TIPS FOR SUCCESS **Experiment 2** **15** *This page intentionally left blank* Name: Date: Section: **Observations and Results Culture \"A**\" Growth (+) or (-) Orange-red pigmentation (+) or (-) Draw the distribution of growth. **Nutrient Broth** **Nutrient Agar Slant** **Nutrient Agar Deep** **Observations and Results Culture** \"**B\"** Growth (+) or (-) Orange-red pigmentation **Nutrient Broth** **Nutrient Agar Nutrient Agar** **Deep** (+) or (-) Draw the distribution of growth. **EXPERIMENT** 2 **Lab Report** **Experiment 2: Lab Report** **17** **Observations and Results** *S. **marcescens*** Growth (+) or (-) Orange-red pigmentation **Nutrient Broth** **Nutrient Agar Slant** **Nutrient Agar Deep** (+) or (-) Draw the distribution of growth. 1\. Explain why the following steps are essential during subculturing: **a.** Flaming the inoculating instrument *prior to and after* each inoculation **b.** Holding the test tube caps in the hand as illustrated in Figure 2.1 on page 14 **c.** Cooling the inoculating instrument prior to obtaining the inoculum **d.** Flaming the neck of the tubes immediately after uncapping and before recapping 2\. Describe the purposes of the subculturing procedure. 3\. Explain why a straight inoculating needle is used to inoculate an agar deep tube. **4.** **5.** 、,, There is a lack of orange-red pigmentation in some of the growth on your agar slant labeled *S. marcescens*. Does this necessarily indicate the presence of a contaminant? Explain. Upon observation of the nutrient agar slant culture, you strongly suspect that the culture is contaminated. Outline the method you would follow to ascertain whether your suspicion is justified. **18 Experiment 2**: **Lab Report** **Techniques for Isolation of Pure Cultures** **EXPERIMENT** 3 In nature, microbial populations do not segregate themselves by species, but exist with a mixture of many other cell types. In the laboratory, we can separate these populations into **pure cultures**. These cultures contain only one type of organism and allow us to study their cultural, morphologi- cal, and biochemical properties. In this experiment, you will first use one of the techniques designed to produce discrete colonies. Colonies are individual, macroscopically visible masses of microbial growth on a solid medium surface, each representing the multiplication of a single organism. Once you have obtained these discrete colonies, you will make an aseptic trans- fer onto nutrient agar slants for the isolation of pure cultures. **PART A** Isolation of Discrete Colonies from a Mixed Culture **LEARNING OBJECTIVE** Once *you* have completed *this experiment, you should* be *able to* 1\. Perform the streak-plate and/or the spread- plate inoculation procedure to separate the cells of a mixed culture so that discrete colonies can be isolated. **Principle** The techniques commonly used for isolation of discrete colonies initially require that the number of organisms in the inoculum be reduced. The resulting diminution of the population size ensures that, following inoculation, individual cells will be sufficiently far apart on the surface of the agar medium to separate the different species. The following are techniques that we can use to accomplish this necessary dilution. 1\. The **streak-plate** method is a rapid qualitative isolation method. It is a dilution technique that spreads a loopful of culture over the surface of an agar plate as a means to separate and dilute the microbes and ensure individual colony growth. There are many different procedures for preparing a streak plate; the four-way, or quadrant, streak will be described. **Figure 3.1** illustrates this technique. surface in Area 1. Flame the loop, cool it by touching it to an unused part of the agar surface close to the periphery of the plate, and then drag it rapidly several times across the surface of Area 1. **b.** Reflame and cool the loop, and turn the Petri dish 90°. Then touch the loop to a corner of the culture in Area 1 and drag it several times across the agar in Area 2. The loop should never enter Area 1 again. **c.** Reflame and cool the loop and again; turn the dish 90°. Streak Area 3 in the same manner as Area 2. Flame loop. **1** Flame loop. Turn plate 90°. Turn plate 90°. **2** Flame loop. **2** **Figure 3.1 Four-way streak-plate technique** **3** **2** **19** Heavy confluent growth Light growth **Figure 3.2 Four-way streak-plate inoculation with *Serratia marcescens*** **d.** Without reflaming the loop, again turn the dish 90° and then drag the culture from a corner of Area 3 across Area 4, using a wider streak. Don\'t let the loop touch any of the previously streaked areas. The purpose of flaming of the loop at the points indicated is to dilute the culture so that fewer organisms are streaked in each area, resulting in the final desired separation. **Figure 3.2** shows a photograph of a streak- plate inoculation. 2\. An alternative streak-plate method is for students new to the laboratory who have yet to master the necessary lab skills that would allow them to use the rapid method listed above. This alternative method involves spreading a loopful of culture over the surface of an agar plate that has the quadrants laid out visibly for quick reference. **Figure 3.3** illustrates this technique. on the bottom of the Petri dish to divide the plate into 4 equal parts. Label each quad- rant 1 through 4, starting with the top right quadrant and labeling counterclockwise. When we sterilize the loop at the indi- cated points, the culture is diluted because fewer organisms are available to streak into each area. This gives us the final desired separation. Petri dish 90°. Then touch the loop into an area that has been streaked in quad- rant 1 and drag it across the agar into quadrant 2. Repeat this twice without flaming the loop. **(a)** Label bottom of dish **(b)** 1 3 Ag и Δε **S** s **E** **3** Δ **ε** L 4 **Figure** 3.3 **Alternate streak-plate method** **20** **Experiment 3** **3** n 4 turn the dish 90°. Streak the bacteria into quadrant 4 in the same manner used for quadrant 3. 3\. The **spread-plate** technique requires that we use a previously diluted mixture of micro- organisms. During inoculation, the cells are spread over the surface of a solid agar medium with a sterile, L-shaped bent glass rod while the Petri dish is spun on a "lazy Susan" turn- table. The step-by-step procedure for this tech- nique is as follows: add a sufficient amount of 95% ethyl alcohol to cover the lower, bent portion. **b.** Place an appropriately labeled nutrient agar plate on the turntable. With a sterile pipette, place one drop of sterile water on the center of the plate, followed by a sterile loopful of *Micrococcus luteus*. Mix gently with the loop and replace the cover. turntable. **e.** While the turntable is spinning, lightly touch the sterile bent rod to the surface of the agar and move it back and forth. This will spread the culture over the agar surface. f\. When the turntable comes to a stop, replace the cover. Immerse the rod in alcohol and reflame. **g.** In the absence of a turntable, turn the Petri dish manually and spread the culture with the sterile bent glass rod. 4\. The **pour-plate** technique requires a serial dilution of the mixed culture by means of a loop or pipette. The diluted inoculum is then added to a molten agar medium in a Petri dish, mixed, and allowed to solid- ify. Experiment 19 outlines the serial dilution and pour-plate procedures. **FURTHER READING** Refer to the section on colony isolation to review other methods beyond the streak-plate technique to isolate microbes and to study colony forma- tion. Use the index to search for the terms \"Streak plate\" and \"Colony.\" CLINICAL APPLICATION **AT THE BENCH** **Materials** **Cultures** 24- to 48-hour nutrient broth cultures of culture from one of the environmental sources listed above. **Media** **Experiment 3** **21** **Equipment** Microincinerator **or** Bunsen burner Inoculating loop Turntable Glassware marking pencil or Sharpie Culture tubes containing 1 ml of sterile water Test tube rack Sterile cotton swabs **Procedure** Lab One 1\. Following the procedures previously described, prepare a spread-plate and/or streak-plate inoculation of each test culture on an appropriately labeled plate. 2\. Prepare an environmental mixed culture. **a.** Dampen a sterile cotton swab with ster- ile water. Wring out the excess water by pressing the wet swab against the walls of the tube. **b.** With the moistened cotton swab, obtain your mixed-culture specimen from one of the selected environmental sources listed in the section on cultures. **c.** Place the contaminated swab back into the tube of sterile water. Mix gently and let stand for 5 minutes. **d.** Perform spread-plate and/or streak-plate inoculation on an appropriately labeled plate. 3\. Incubate all plates in an **inverted position** for 48 to 72 hours at 25°C. **Procedure** Lab Two 1\. Examine all agar plate cultures to identify the distribution of colonies. In the charts provided in Part A of the Lab Report, complete the following: **a.** Draw the distribution of colonies appearing on each of the agar plate cultures. **b.** On each of the agar plate cultures, select two discrete colonies that differ in appear- ance. Using Figure 4.1 on page 30 as a reference, describe each colony\'s Form: circular, irregular, or spreading Pigmentation Size: pinpoint, small, medium, **or** large. **TIPS** FOR SUCCESS **1. An isolation plate has isolated distinct**, **indi-** **vidual colonies.** If your technique results in isolated colonies in a quadrant that was not the last one to be streaked, that is okay. The point of using this method is to get those individual colo- nies somewhere on the plate. **2. Pay attention to how well you sterilize your** **PART B** Isolation of Pure Cultures from a Spread-Plate or Streak-Plate Preparation *Once you* have *completed this experiment, you should be* able *to* 1\. Prepare a stock culture of an organism using isolates from mixed cultures prepared on an agar streak plate and/or spread plate. **Principle** Once discrete, well-separated colonies develop on the surface of a nutrient agar plate culture, each may be picked up with a sterile needle and transferred to separate nutrient agar slants. Each of these new slant cultures represents the growth of a single bacterial species and is designated as a **pure culture** or **stock culture**. **22** **Experiment 3** **Transferring a Colony of Bacteria Daughter Cells** **Equipment** Microincinerator or Bunsen burner Inoculating needle Glassware marking pencil **Procedure** Lab One 1\. Aseptically transfer, from visibly discrete colo- nies, the yellow *M. luteus,* the white *E. coli,* the red *S. marcescens*, and a discrete colony from the environmental agar plate specimen to the appropriately labeled agar slants as shown in **Figure 3.4**. **AT** THE **BENCH** **Materials** **Cultures** Mixed-culture, nutrient agar streak-plate and/or spread-plate preparations of *S. marcescens* and *M. luteus M. luteus* and *E. coli* Environmental specimen plate from Part A **Media** Four Trypticase soy agar slants per designated student group **Procedure** Lab Two 1\. In the chart provided in Part B of the Lab Report, complete the following: **a.** Draw and indicate the type of growth of **b.** Observe the color of the growth and record its pigmentation. **Experiment 3** **23** **PROCEDURE** **1** Flame the straight needle until the entire wire is red. **2** After isolating a discrete colony on the agar streak plate, touch the straight needle to the surface of the selected colony. **3** Uncap the agar slant and pass the neck of the tube rapidly over the Bunsen burner flame. 4 Inoculate the slant by drawing the needle upward in a zigzag motion along the surface of the agar. Do not dig into the agar. **6** Flame the inoculating needle. **5** Flame the neck of the tube and recap. **Figure 3.4 Procedure for the preparation of a pure culture** **24** **Experiment 3** Name: Date: **Observations and Results** **PART A: Isolation of Discrete Colonies** **from a Mixed Culture** Draw the colonies that appear on each agar plate. **EXPERIMENT** 3 **Lab Report** S. ***marcescens* and *M. luteus*** **M. *luteus* and *E. coli*** Colony description: Isolate 1 Isolate 2 Isolate 3 Isolate 4 Form Pigmentation Size Draw the colonies that appear on each agar plate. Colony description: Form Elevation Pigmentation Size ENVIRONMENTAL SPECIMEN **Streak-Plate Technique** **PART B: Isolation of Pure Cultures from a Spread-Plate** **or Streak-Plate Preparation** Draw the distribution of growth on the slant surface. Type of growth Pigmentation Name of organism 26 **Experiment 3: Lab Report** **Review Questions** 1\. Can you prepare a pure culture from a mixed-broth or a mixed-agar-slant culture? Explain. 2\. Observation of a streak-plate culture shows more growth in quadrant 4 than in quadrant 3. Account for this observation. **3.** Why is a needle used to isolate individual colonies from a spread plate or 4\. How can you determine if the colony that you chose to isolate is a pure **Experiment 3: Lab Report** **27** *This page intentionally left blank* **EXPERIMENT** 4 **LEARNING OBJECTIVE** Once *you* have *completed* this *experiment, you should be able to* **1.** Determine the cultural characteristics of microorganisms as an aid to identify and classify organisms into taxonomic groups. **Principle** When grown on a variety of media, microorgan- isms exhibit differences in the macroscopic appearance of their growth. We use these differences, called **cultural characteristics**, to separate microorganisms into taxonomic groups. The *Bergey\'s Manual of Systematic Bacteriology* outlines the cultural characteristics for all known microorganisms. They are determined by culturing the organisms on nutrient agar slants and plates, in nutrient broth, and in nutrient gelatin. The patterns of growth in each of these media are described below, and some are illustrated in **Figure 4.1**. **Nutrient Agar Slants** These have a single straight line of inoculation on the surface and are evaluated by 1\. **Abundance of growth:** The amount of growth is designated as none, slight, moderate, **or** large. 2\. **Pigmentation:** Chromogenic microorganisms may produce intracellular pigments that are responsible for the coloration of the organ- isms as seen in surface colonies. Other organ- isms produce extracellular soluble pigments that are excreted into the medium and also produce a color. Most organisms, however, are nonchromogenic and will appear white to gray. 3\. **Optical characteristics:** Optical charac- teristics may be evaluated by the amount of light transmitted through the growth. These characteristics are **opaque** (no light transmis- sion), **translucent** (partial transmission), or **transparent** (full transmission). **4. Form:** The appearance of the single-line streak of growth on the agar surface is desig- nated as with smooth edges **b. Echinulate:** continuous, threadlike growth with irregular edges 5\. **Consistency:** **c. Mucoid:** slimy and glistening **Nutrient Agar Plates** 1\. **Size:** pinpoint, small, moderate, or large 2\. **Pigmentation:** color of colony **3.** **Form:** The shape of the colony is described as follows: 4\. **Margin:** The appearance of the outer edge of the colony is described as follows: **a. Entire:** sharply defined, even **b. Lobate:** marked indentations **c. Undulate:** wavy indentations **d. Serrate:** toothlike appearance **5.** **a. Flat:** elevation not discernible **b. Raised:** slightly elevated **29** Crateriform Napiform Entire Circular Infundibuliform **(a)** Gelatin liquefaction Forms Saccate Stratiform Rhizoid Flat Irregular Undulate Lobate Margins Convex Serrate Filamentous Umbonate Elevation DUB B Filiform Echinulate Beaded Effuse **(d)** Growth on agar slants **Figure 4.1 Cultural characteristics of bacteria** **30 Experiment 4** Uniform fine turbidity Flocculent growth I Pellicle I **(c)** Growth in broth media Arborescent Rhizoid **Nutrient Broth Cultures** 1\. **Uniform fine turbidity:** finely dispersed throughout 3\. **Pellicle:** thick, padlike growth on surface 4. **Sediment:** Concentration of growth at the bottom of broth culture may be granular, flaky, **or** flocculent. **Nutrient Gelatin** This solid medium may be liquefied by the enzy- matic action of gelatinase. Liquefaction occurs in a variety of patterns: 1\. **Crateriform:** Liquefied surface area is saucer-shaped. 2\. **Napiform**: Bulbous-shaped liquefaction at 3\. **Infundibuliform:** Funnel-shaped 4\. **Saccate:** Elongated, tubular 5\. **Stratiform:** Complete liquefaction of the **FURTHER READING** Refer to your textbook for description and expla- nations of growth characteristics that will lead to the different colony morphologies seen in this experiment. In your textbook\'s index, use the search terms \"Colony,\" \"Pigmentation," and \"Growth Curve.\" CLINICAL APPLICATION **Examining Colony Growth Characteristics to Aid Identification** Bacterial species each have a characteristic pattern of colony growth in a liquid culture or on a solid medium. While not truly a diagnostic tool, our recognition of these characteristic patterns in a clinical lab setting helps us minimize the list of potential bacterial species to test for. **AT** THE **BENCH** **Materials** **Cultures** Twenty-four-hour nutrient broth cultures of *Pseudomonas aeruginosa* **BSL-2** *Bacillus cereus* *Micrococcus luteus* *Escherichia coli* 72-to-96-hour Trypticase® soy broth culture of *Mycobacterium smegmatis* **Media** Nutrient gelatin tubes **Equipment** Microincinerator or Bunsen burner Inoculating loop and needle Glassware marking pencil **Procedure** Lab One 1\. Using aseptic technique, inoculate each of the appropriately labeled media in the following list: **b.** Nutrient agar plates: With a sterile loop, prepare a streak-plate inoculation of each of the cultures for the isolation of discrete colonies. **c**. Nutrient broth cultures: Using a sterile loop, inoculate each organism into a tube of nutrient broth. Shake the loop a few times to dislodge the inoculum. **d.** Nutrient gelatin: Using a sterile needle, prepare a stab inoculation of each culture provided. **Experiment 4** **31** **Procedure** Lab Two 1\. Before beginning observation of all the cultures, place the gelatin cultures in a refrig- erator for 30 minutes or in a beaker of crushed ice for 5 to 10 minutes. Observe the gelatin culture last. 2\. Refer to Figure 4.1 on page 30 and this Experiment\'s introduction while observing the following: **a.** Nutrient agar slants: Observe each of the nutrient agar slant cultures for the amount, pigmentation, form, and consistency of the growth. Record your observations in the chart provided in the Lab Report. **b.** Nutrient agar plates: Observe a single, well-isolated colony on each of the nutrient agar plate cultures and identify its size, elevation**,** margin, form, and pigmenta- tion. Record your observations in the chart provided in the Lab Report. **c.** Nutrient broth cultures: Observe each of the nutrient broth cultures for the appearance of growth (flocculation, turbidity, sediment, **or** pellicle). Record your observations in the chart provided in the Lab Report. from the refrigerator or beaker of crushed ice, and observe whether liquefaction of the medium has developed and whether the organism has produced gelatinase. Record your observations in the chart provided in the Lab Report. **32 Experiment 4** Name: Date: **Observations and** Results **Nutrient Agar Slants** Draw the distribution of growth on the slant surface. Amount of growth Pigmentation Form Consistency NUTRIENT AGAR SLANT CULTURES ***M. luteus*** **P. *aeruginosa M. smegmatis*** **Nutrient Agar Plates** Draw distribution of colonies. Size Elevation Margin Form Pigmentation **EXPERIMENT** 4 **Lab Report** **E. *coli*** ***M. luteus*** **P. *aeruginosa*** **M. *smegmatis*** **E. *coli*** ***B.*** **33** **Nutrient Broth Cultures** Draw the distribution of growth. Appearance of growth **Nutrient Gelatin** Draw liquefaction patterns. Liquefaction (+) or (-) Type of liquefaction NUTRIENT BROTH CULTURES ***M. luteus*** **P. *aeruginosa*** **M. *smegmatis*** NUTRIENT GELATIN CULTURES **34** **Experiment 4: Lab Report** **M. *smegmatis*** **E. *coli*** **B. *cereus*** ***E. coli*** ***B.* cereus**