Information

Lab 15: Isolation and Identification of Staphylococci - Biology


DISCUSSION

Staphylococci (see Fig. 6) are often found in the human nasal cavity (and on other mucous membranes) as well as on the skin. They are Gram-positive cocci 0.5-1.0 µm in diameter and occur singly, in pairs, in short chains, and most commonly, in irregular grape-like clusters. The staphylococci are strongly catalase positive and generally tolerate relatively high concentrations of sodium chloride (7.5-10%). This ability is often employed in preparing media selective for staphylococci.

Staphylococcal capsules play a major role in the ability of the bacteria to adhere to and colonize biomaterials.

  • Scanning electron micrograph Staphylococcus aureus; courtesy of Dennis Kunkel's Microscopy.
  • Scanning electron micrograph of methicillin-resistant Staphylococcus aureus (MRSA); courtesy of CDC
  • Scanning electron micrograph of methicillin-resistant Staphylococcus aureus (MRSA) destroying an immune cell; courtesy of Frank Deleo, NIAID

There are five species of staphylococci commonly associated with clinical infections: Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. hominis and S. saprophyticus.

A. Staphylococcus aureus (coagulase-positive staphylococci)

Staphylococcus aureus is the most pathogenic species and is implicated in a variety of infections. S. aureus is with some frequency found as normal human flora in the anterior nares (nostrils). It can also be found in the throat, axillae, and the inguinal and perineal areas. Approximately 30% of adults and most children are healthy periodic nasopharyngeal carriers of S. aureus. Around 15% of healthy adults are persistent nasopharyngeal carriers. The colonization rates among health care workers, patients on dialysis, and people with diabetes are higher than in the general population.

In the majority of S. aureus infections the source of the organism is either:

  • a healthy nasal carrier, or
  • contact with an abscess from an infected individual.

The portal of entry is usually the skin. aureus causes pus-filled inflammatory lesions known as abscesses. Depending on the location and extent of tissue involvement, the abscess may be called:

1. a pustule
A pustule is an infected hair follicle where the base of the hair follicle appears red and raised with an accumulation of pus just under the epidermis. Infected hair follicles are also referred to as folliculitis.

2. a furuncle or boil
Furuncles appear as large, raised, pus-filled, painful nodules having an accumulation of dead, necrotic tissue at the base. The bacteria spread from the hair follicle to adjacent subcutaneous tissue.

3. a carbuncle
Carbuncles occur when furuncles coalesce and spread into surrounding subcutaneous and deeper connective tissue. Superficial skin perforates, sloughs off, and discharges pus.

S. aureus also causes impetigo, a superficial blister-like infection of the skin usually occuring on the face and limbs and seen mostly in young children. aureus may also cause cellulitis, a diffuse inflammation of connective tissue with severe inflammation of dermal and subcutaneous layers of the skin. aureus is also a frequent cause of accidental wound and postoperative wound infections.

Less commonly, S. aureus may escape from the local lesion and spread through the blood to other body areas, causing a variety of systemic infections that may involve every system and organ. Such systemic infections include septicemia, septic arthritis, endocarditis, meningitis, and osteomyelitis, as well as abscesses in the lungs, spleen, liver, and kidneys. S. aureus pneumonia may also be a secondary respiratory complication of viral infections such as measles, and influenza and is a frequent cause of nosocomial pneumonia in patients who are debilitated. Finally, S. aureus is frequently introduced into food by way of abscesses or the nasal cavity of food handlers. If it is allowed to grow and produces an enterotoxin, it can cause staphylococcal food poisoning.

In a 1990-1992 National Nosocomial Infections survey, CDC found S. aureus to be the most common cause of nosocomial pneumonia and operative wound infections, as well as the second most common cause of nosocomial bloodstream infections. Antibiotic resistant S. aureus is a common problem. For example, a survey conducted by CDC reported the proportion of methicillin-resistant isolates S. aureus (MRSA) with sensitivity only to vancomycin increased from 22.8% in 1987 to 56.2% in 1997.

Virulence factors for S. aureus include exotoxins such as leukocidin (kills leukocytes), alpha and delta toxins (damage tissue membranes), microcapsules (resist phagocytic engulfment and destruction), coagulase and protein A (both help resist phagocytic engulfment). Some strains also produce TSST-1 (toxic shock syndrome toxin-1) and cause toxic shock syndrome, usually associated with wounds. Approximately 25% of all S. aureus strains are toxigenic and approximately 6000 gases of toxic shock syndrome occur each year in the U.S. Some strains also produce exfoliatin, an exotoxin that causes scalded skin syndrome, an infection usually seen in infants and young children.

For further information on virulence factors associated with S. aureus, see the following Learning Objects in your Lecture Guide:

  • Teichoic Acids and Glycopeptides Cell Wall Fragments; Unit 3, Section C1c
  • The Ability to Adhere to Host Cells: Pili and Adhesins; Unit 3, Section B2
  • The Ability to Resist Phagocytic Engulfment; Unit 3, Section B5b
  • The Ability to Resist Phagocytic Destruction and Serum Lysis; Unit 3, Section B5c
  • The Ability to Evade Adaptive Immune Defenses; Unit 3, Section B6
  • The Ability to Produce Harmful Superantigens; Unit 3, Section C2a1
  • A-B Toxins; Unit 3, Section C2b
  • Toxins that Damage Cell Membranes; Unit 3, Section C2c

Since most S. aureus strains produce the enzyme coagulase (see the coagulase test described below), they are often referred to as coagulase-positive staphylococci.

  • Scanning electron micrograph of Staphylococcus aureus forming a biofilm in an indwelling catheter.
  • Diseases and Organisms in Healthcare Settings; from CDC

B. Coagulase-Negative Staphylococci

Clinically common species of staphylococci other than S. aureus are often referred to as coagulase-negative staphylococci. These staphylococci are normal flora of the skin and, as such, frequently act as opportunistic pathogens, especially in the compromised host. saprophyticus is a relatively common cause of urinary tract infections, especially in healthy young women, but is seldom isolated from other sources. The great majority of infections caused by other coagulase-negative staphylococci, including S. haemolyticus, and S. hominis, are associated with intravascular devices (prosthetic heart valves and intra-arterial or intravenous lines) and shunts. Also quite common are infections of prosthetic joints, wound infections, osteomyelitis associated with foreign bodies, and endocarditis.

  • Electron micrograph of Staphylococcus epidermidis colonizing a vascular catheter.
  • Diseases and Organisms in Healthcare Settings; from CDC

Although certain reactions may vary from strain to strain, a series of biochemical tests will usually differentiate the most common clinically encountered species of staphylococci. Today we will use a number of tests to determine if an unknown is S. aureus, S. epidermidis, or S. saprophyticus.

ISOLATION AND IDENTIFICATION OF STAPHYLOCOCCI

1. Blood agar with a novobiocin (NB) disc

To isolate staphylococci, clinical specimens are usually grown on Blood agar (described in Lab 14). Staphylococci produce round, raised, opaque colonies 1-2mm in diameter. The novobiocin disc is used to detect sensitivity or resistance to the antibiotic novobiocin.

Test

Staphylococcus aureus,
pigmented strain

S. aureus,
non-pigmented strain

Staphylococcus epidermidisStaphylococcus saprophyticus
Hemolysis (*)Usually beta(1)Usually gamma(2)Usually gamma(2)
PigmentOften creamy gold(1)Usually white(2)Usually white(2)
Novobiocin testSensitiveSensitiveResistant

(*) see Lab 14 for descriptions of hemolysis
(1) some strains do not show hemolysis and/or pigment
(2) some strains do show hemolysis and/or pigment
sensitive = zone of inhibition around disc
resistant = no zone of inhibition around disc

2. Gram stain

All staphylococci appear as Gram-positive cocci, usually in irregular, often grape-like clusters (see Fig. 6).

3. Mannitol fermentation on Mannitol Salt agar (MSA)

Staphylococci are able to tolerate the high salt concentration found in Mannitol Salt agar and thus grow readily. If mannitol is fermented, the acid produced turns the phenol red pH indicator from red (alkaline) to yellow (acid).

positive = acid end products turn the phenol red pH indicator from red to yellow
negative = prenol red remains red

4. Production of deoxyribonuclease (DNase) on DNase agar

DNase agar contains 0.2% DNA. To detect DNase production, the plate is inoculated and incubated. After growth, the plate is flooded with 1N hydrochloric acid (HCl). DNase positive cultures show a distinct clear zone around the streak (see Fig. 1) where the DNA in the agar was broken down by the bacterial DNase. DNase negative cultures appear cloudy around the growth where the acid caused the DNA in the agar to precipitate out of solution.

positive = clear zone around growth after adding 1N HCl (no DNA remaining in the agar)
negative = cloudy around growth after adding 1N HCl (DNA remains in the agar forming a precipitate)

5. Production of coagulase

The staphylococcal enzyme coagulase will cause inoculated citrated rabbit plasma to gel or coagulate. The coagulase converts soluble fibrinogen in the plasma into insoluble fibrin.

positive = plasma will gel or coagulate
negative = plasma will not gel

6. The Staphyloslide® Latex Test for cell-bound coagulase (clumping factor) and/or Protein A

The Staphyloslide® Latex Test is an agglutination test that detects cell-bound coagulase (clumping factor) and/or Protein A. Approximately 97% of human strains of S. aureus possess both bound coagulase and extracellular coagulase. Approximately 95% of human strains of S. aureus possess Protein A on their cell surface (see Fig. 7). This test uses blue latex particles coated with human fibrinogen and the human antibody IgG. Mixing of the latex reagent with colonies of the suspected S. aureus having coagulase and/or Protein A bound to their surface causes agglutination of the latex particles.

positive = clumping of latex particles
negative = no clumping of latex particles

For further information on coagulase and Protein A associated with S. aureus, see the following Learning Objects in your Lecture Guide:

  • The Ability to Resist Phagocytic Engulfment; Unit 3, Section B5b
  • The Ability to Evade Adaptive Immune Defenses; Unit 3, Section B6

Staphylococci are also being identified using chemiluminescent labelled DNA probes complementary to species-specific bacterial ribosomal RNA (rRNA) sequences as well as by other direct DNA techniques.

SCENERIO FOR TODAY'S LAB

Choose either unknown #1 or unknown #2 as your unknown for this Case Study.

A 57 year old female who is diabetic, a smoker, and who 30 days ago had hip replacement surgery presents with pain and signs of inflammation at the surgical site. Examination shows she has a fever of 101 °F and an increased total white blood cell count with a left shift. Radiologic examination shows a deep pelvic abcess. A culture of the implant site was taken.

Assume that unknown you are given is the culture from this patient.

MATERIALS

1 plate of blood agar, 1 novobiocin (NB) disc, 1 plate of mannitol salt agar, 1 DNase agar plate, 1 tube of citrated rabbit plasma (coagulase test), materials to perform a Gram stain, inoculating loop

PROCEDURE (to be done in groups of 3)
[Keep in mind that in a real clinical situation other lab tests and cultures for bacteria other than those upon which this lab is based would also be done.]

CAUTION: TREAT EACH UNKNOWN AS A PATHOGEN!. Inform your instructor of any spills or accidents. WASH AND SANITIZE YOUR HANDS WELL before leaving the lab.

1. Do a Gram stain on the unknown (see Lab 6). Make sure you review the instructions before you do the Gram stain.

2. Using your loop, streak your unknown for isolation on a plate of Blood agar as described below.

a. Using a sterile inoculating loop, streak your unknown for isolation on a blood agar plate so as to get single, isolated colonies (Fig. 2, step 1, Fig. 2, step 2, and Fig. 2, step 3). Before you streak your plate draw an "X" on the bottom of the blood agar plate to indicate where you begin the streaking pattern.

b. Using your inoculating loop, stab the agar several times in each of the growth areas in order to detect oxygen-sensitive hemolysins (Fig. 2, step 4).

c. Place a novobiocin antibiotic disc in the center of the area of the plate that you streaked first (the area where you drew the "X") where you expect to see heaviest growth (Fig. 2, step 5).

d. Incubate upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section until the next lab period.

3. Streak your unknown for isolation on a plate of Mannitol Salt agar (MSA) as shown in Fig. 3. Incubate upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section until the next lab period.

4. Streak a single line of your unknown down the center of a plate of DNase agar as shown in Fig. 4. Incubate upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section until the next lab period.

5. Inoculate a tube of citrated rabbit plasma with your unknown and incubate your test tube rack at 37°C.

RESULTS

Case Study Lab Report for Lab 15:
Staphylococci

The concept behind the case studies presented in Lab 15 used to illustrate the genus Staphylococcus is for you and your lab partners as a group to:

1. Come up with a valid diagnosis of the type of infectious disease seen in your case study and identify the bacterium causing that infection.

2. Support your group’s diagnose based on:

a. Any relevant facts in the patient’s history. (A reliable on-line source will be used to support this.)
b. The patient’s signs and symptoms. (A reliable on-line source will be used to support this.)
c. Each of the individual lab tests given in your case study.
d. All microbiological lab tests you performed as part of the project.

The due date for this report can be found on the class calendar. Remember, you are working as a group to solve a problem. Your grade for this lab is based on the completeness of your report and written evidence of the critical thinking process that went into making and supporting your diagnosis, therefore, it is critical that all members of the group participate, question any conclusions being made by the group, and contribute to the report. Remember, you are trying to convince your instructor that you understand how the diagnosis was made by supporting that diagnosis with data. Your group will work together to write the report and submit one hard copy of that report for your group. Part of your grade will be based on evaluation of your work by your team members.

Be sure to handle all the bacterial cultures you are using in lab today as if they are pathogens! Be sure to wash and sanitize your hands well at the completion of today’s lab.

Also, make sure you observe the results of someone in your lab who had an unknown different from yours . The Performance Objectives for Lab 15 tell you what you are expected to be able to do on the practical.

Each member of the group must:

1. Print a copy of each of the two rubrics from the links above.

2. Print and fill out a copy of the Team Member Evaluation Form from the link above.

3. Staple them together and hand them in to me the day your Lab 15 Case Study Lab Report is due.

For more information on writing your Lab Report, see Course evaluation for Lab (Core labs, case studies, lab quizzes) under Course Info in the menu of the BIOL 230 website.

Click here to print a PDF copy of this lab report.

Your Name:

Others in your group:

Lab section:

Date:

A. Case Study from Lab 15: Unknown #1

Each member of the group must:

1. Staple them together and hand them in to me the day your Lab 15 Case Study Lab Report is due.

A 57 year old female who is diabetic, a smoker, and who 30 days ago had hip replacement surgery presents with pain and signs of inflammation at the surgical site. A culture of the implant site was taken.

1. Patient’s history and predisposing factors

Read the case study. Explain how any relevant parts of the patient’s history contributed to your diagnosis as to the type of infectious disease seen here. You are urged to use the computers in lab to search reliable medically oriented Internet sources to support this. Reliable sources you might consider are Medscape (http://emedicine.medscape.com/infectious_diseases) and The Centers for Disease Control and Prevention (CDC) at http://www.cdc.gov/. Cite any sources you use at the end of this Patient's History section in APA style (http://www.apastyle.org/).

The patient's history should suggest a general type of infectious disease that is present, such as a urinary tract infection, a wound infection, gastroenteritis, pharyngitis, pneumonia, septicemia, etc. Do not look up the bacterium you eventually identify as the cause of this infectious disease. You don't know the causative bacterium at this point. You need to determine the general type of infection in order to determine what microbiological tests to perform to identify the bacterium causing the infection. Search at least one medically-oriented reference article from a reliable site such as Medscape and use this article to support your diagnosis of the type of infectious disease seen here. Don't forget to cite any sources you used in APA style directly under this Patient's History and Patient's Symptoms sections of this Lab Report.

2. Patient’s signs and symptoms

Read the case study. Explain how the patient’s signs and symptoms contributed to your diagnosis as to the type of infectious disease seen here. Reliable sources you might consider are Medscape (http://emedicine.medscape.com/infectious_diseases)
and The Centers for Disease Control and Prevention (CDC) at http://www.cdc.gov/. Cite any sources you use at the end of this Patient's Symptoms section in APA style (http://www.apastyle.org/).

The patient's signs and symptoms should suggest a general type of infectious disease that is present, such as a urinary tract infection, a wound infection, gastroenteritis, pharyngitis, pneumonia, septicemia, etc. Don't forget to cite any sources you used in APA style directly under this Patient's History and Patient's Symptoms sections of this Lab Report.

3. Results of laboratory test given in the case study

List each lab test given and explain how the results of that test helps to contribute to your diagnosis. The CBC test is described in Appendix C of this lab manual.

4. Microbiological lab tests you performed in Lab 15

a. Gram stain

Give the Gram reaction (Gram-positive or Gram negative and how you reached this conclusion) and the shape and arrangement of the unknown you were given. State how this contributed to your decision as to which microbiological tests and/or media to use next. The Gram stain is discussed in Lab 6.

b. Blood agar with novobiocin (NB) disc

Give the results of the Blood agar with Taxo NB disc you performed on the unknown you were given, and how you reached this conclusion.State how this contributed to your your decision as to what bacterium is causing the infection. The possible results for Blood agar and NB disc were discussed in the beginning pages of this lab.

c. Mannitol Salt agar

Give the results of the Mannitol Salt agar you performed on the unknown you were given, and how you reached this conclusion.State how this contributed to your your decision as to what bacterium is causing the infection. The possible results for Mannitol Salt agar were discussed in the beginning pages of this lab.

d. DNase agar

Flood the surface of your DNase agar plate with 1N HCl. Give the results of the DNase agar you performed on the unknown you were given, and how you reached this conclusion.State how this contributed to your your decision as to what bacterium is causing the infection. The possible results for DNase agar were discussed in the beginning pages of this lab.

e. Coagulase test

Give the results of the Coagulase test you performed on the unknown you were given, and how you reached this conclusion.State how this contributed to your your decision as to what bacterium is causing the infection. The possible results for the Coagulase test were discussed in the beginning pages of this lab.

Final Diagnosis

Genus and species of unknown #1 = ________________________________

Infection: _______________________________

B. Case Study from Lab 15: Unknown #2

Each member of the group must:

1. Explain how the patient’s symptoms contributed to your diagnosis as to the type of infectious disease seen here. Don't forget to cite any sources you used in APA style directly under this Patient's History and Patient's Symptoms sections of this Lab Report.

3. The CBC test is described in Appendix C of this lab manual.

4. Microbiological lab tests you performed in Lab 15

a. Blood agar with novobiocin (NB) disc

Give the results of the Blood agar with Taxo NB disc you performed on the unknown you were given, and how you reached this conclusion.State how this contributed to your your decision as to what bacterium is causing the infection. The possible results for Blood agar and NB disc were discussed in the beginning pages of this lab.

Give the results of the Mannitol Salt agar you performed on the unknown you were given, and how you reached this conclusion.State how this contributed to your your decision as to what bacterium is causing the infection. The possible results for Mannitol Salt agar were discussed in the beginning pages of this lab.

d. The possible results for the Coagulase test were discussed in the beginning pages of this lab.

Final Diagnosis

Genus and species of unknown #2 = ________________________________

Infection: _________________________________

PERFORMANCE OBJECTIVES FOR LAB 15

After completing this lab, the student will be able to perform the following objectives:

DISCUSSION

1. Name three common clinically important species of Staphylococcus and state which species is most pathogenic.

2. State the sources and the portal of entry for most Staphylococcus aureus infections.

3. Name and describe three types of abscesses caused by Staphylococcus aureus.

4. Name four systemic Staphylococcus aureus infections.

5. State the significance of Staphylococcus aureus enterotoxin, the exotoxin TSST-1, and the exotoxin exfoliatin.

6. Name the infection normally caused by Staphylococcus saprophyticus.

7. Name the types of infections most commonly caused by coagulase-negative staphylococci other than Staphylococcus saprophyticus.

ISOLATION AND IDENTIFICATION OF STAPHYLOCOCCI

1. State the Gram reaction and morphology of all staphylococci.

2. Describe the typical reactions of S. epidermidis, and S. saprophyticus on each of the following media:

a. Blood agar (pigment, hemolysis, novobiocin resistance)

b. Mannitol Salt agar (for mannitol fermentation)

c. DNase agar (for the enzyme DNase)

d. coagulase test with citrated rabbit plasma

e. Staphyloslide® test for bound coagulase and/or Protein A

RESULTS

1. Recognize staphylococci in a Gram stain preparation.

2. Recognize an organism as Staphylococcus aureus and state the reasons why after seeing the results of the following:

a. a Blood agar plate with a novobiocin disc

b. a Mannitol Salt agar plate

c. a DNase agar plate

d. a tube of citrated rabbit plasma

e. a Staphyloslide® test

SELF-QUIZ


CDC Streptococcus Laboratory

CDC&rsquos Streptococcus Laboratory provides support for active population-based surveillance for invasive streptococcal disease, primarily caused by groups A and B streptococci and Streptococcus pneumoniae. It supports state and local health departments in the United States to characterize streptococcal isolates and is active in many international collaborations. The laboratory is a reference center for the identification and characterization of streptococci and other Gram-positive catalase-negative cocci.

M protein gene (emm) typing, databases, resources, and protocols.

MICs predicted by penicillin binding protein gene types, resources, protocols, and the global pneumococcal strain bank.

PCR serotyping methods and resources.

Methods for identifying other species and types of streptococci, including PCR assays.

Frequently asked questions about the Streptococcus Laboratory&rsquos reference services and request forms.

Papers published by the Streptococcus Laboratory.

Opportunities concerning molecular epidemiology and biology at the Streptococcus Laboratory.

Group A streptococci: Submit emm gene sequence to determine emm subtype of your strain.

There are many species and subspecies within the Streptococcus genus. CDC provides protocols for how to identify and differentiate species of streptococci.


INTRODUCTION

Using Koch’s postulates for the identification of pathogenic microbes, Ogston identified the etiological agent of suppurative abscesses (Ogston, 1883). The name Staphylococcus aureus was chosen to distinguish this species with its characteristic yellow colony pigment from another staphylococcal commensal that forms white colonies (Staphylococcus albus, now designated Staphylococcus epidermidis) (Rosenbach, 1884 Götz et al., 2006). S. aureus displays several striking microbiological properties, e.g., the microbe binds immunoglobulins and agglutinates with or coagulates blood and plasma (Loeb, 1903 Much, 1908 Forsgren and Sjöquist, 1966 Cheng et al., 2011). These traits have been useful for the early and rapid diagnosis of S. aureus infections (for a historical account of the coagulase test, follow the link http://www.microbelibrary.org/index.php/library/laboratory-test/3220-coagulase-test-protocol).

All Staphylococci grow in clusters, a feature that can be visualized by microscopy and accounts for the Greek name σταΦυλoκoκκoς or grape-like berry. Clustering is caused by the incomplete separation of daughter cells following division in three alternating perpendicular planes (Tzagoloff and Novick, 1977 Giesbrecht et al., 1998). S. aureus cells appear perfectly spherical with a diameter of

1 μm (Giesbrecht et al., 1998).S. aureus also produces catalase when applied to colony material, the catalase test is a rapid, useful test to distinguish staphylococci from other Gram-positive bacteria such as streptococci.

S. aureus is a facultative anaerobe that grows by aerobic respiration or by fermentation, which yields principally lactic acid. The bacterium metabolizes glucose via the pentose phosphate pathway (Reizer et al., 1998). There is no evidence for the existence of the Entner-Doudoroff pathway however, enzymes of the entire tricarboxylic acid cycle and a typical F0F1-ATPase are encoded by the genome of S. aureus (Kuroda et al., 2001). Upon glucose depletion, S. aureus cells growing in aerobic conditions oxidize D-galactose, acetate, succinate and malate. An excellent summary of these metabolic pathways was recently published (Götz et al., 2006).

CAUTION: S. aureus is a highly virulent and adaptable pathogen with the ability to infect, invade, persist, and replicate in any human tissue including skin, bone, visceral organs, or vasculature (Lowy, 1998). The organism has been placed in Risk Group Level 2. All manipulations with S. aureus strains must be performed following biosafety level 2 measures including experimental work in certified biosafety cabinets. Guidelines for BSL2 practice can be obtained from the latest edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL, 5th Edition) via the following CDC Web link: http://www.cdc.gov/biosafety/publications/bmbl5/.


Isolation Methods of Bacteria

The bacterial isolation includes the following plating methods:

Pouring Method

Pouring is the simplest method for bacterial isolation. Here, the bacterial suspension laden with a huge bacterial population is generally taken.

By using a pipette, take 1 ml of a bacterial sample into the sterile Petri plate. For the growth of bacteria, there should be some nutrient source like carbon and nitrogen. Therefore, the most common nutrient agar medium is first prepared and added to the Petri plates containing the bacterial sample.

For the uniform distribution of the sample and the media, rotate the plates in the clockwise and anticlockwise direction. Before keeping the Petri plates in the incubator, allow the culture plates to solidify. For the proper growth of bacteria, keep the culture plates in the incubator at the temperature of 35-37 degrees Celsius for the maximum period of 48 hours. After incubation, we can see the growth of bacterial colonies.

In the pouring method, bacteria’s isolation becomes difficult because of the suspended bacterial growth in the solid media. Some bacteria appear on the surface of the solid nutrient medium, and some appear under the surface of a solid nutrient medium. Pouring method generally results in the overgrowth of bacterial colonies due to which the isolation of pure culture is challenging. This technique is least recommended for obtaining a pure culture.

Spreading Method

Spreading method is again a very simple method to perform bacterial isolation. It slightly differs from the pour plate method. Here, the nutrient medium is added to the Petri plates before adding the bacterial sample. The nutrient medium added to the sterile Petri plates is then allowed to solidify. After solidifying the nutrient medium, Add 1 ml of the bacterial suspension over the medium’s surface.

For the uniform distribution of bacteria over the surface of solid media, take the spreader of T or L-shape to spread the bacterial suspension evenly. After that, incubate the culture plates at 35-37 degrees Celsius for 24 to 48 hours. We can see several bacterial colonies after the incubation. In the spreading method, we can select the isolated colonies for the culturing of bacteria. For the isolation of pure culture, the spreading method is not very popular.

Streaking Method

Streaking method is very popular and the most widely used method for the isolation of pure culture. To perform streaking, pour freshly prepared nutrient agar medium into the sterile Petri plates and allow it to solidify.

After that, take an inoculating loop and sterilize it on the flame until it gets red hot. Then, take the inoculum by using sterilized inoculating loop and streak over the solid nutrient media by keeping the plate close to the flame to avoid contamination. After streaking, incubate the culture plates for 24-48 hours at a temperature of 35-37 degrees Celsius within the incubator.

Due to a limited population of bacteria in the streaking method, pure culture isolation is quite easier than the pour plate and the spread plate method. Through the streaking method, we can culture, isolate and study the individual colony of bacteria.

Serial Dilution Method

This technique is much known for the isolation and culturing of bacteria. In the serial dilution method, take the bacterial suspension and dilute it serially in the successive test tubes.

By following the serial dilution, add 1 ml of the sample to the neighbouring test tube sequentially in a series 10 -1 , 10 -2 , 10 -3 and so on. After the sequential dilution of bacterial suspension, we can inoculate the bacterial culture by using one of the three methods (pouring, spreading and streaking). It is very easy to isolate bacteria from a little bacterial population.

In serial dilution, the more concentrated sample (10 -1 ) will produce the highest number of colonies. The more diluted sample (10 -4 ) will produce the least number of colonies. So it should be clear to us that the less diluted sample will contain more bacteria concentration than water. And the more diluted sample will contain a high concentration of water than that of bacteria.

Therefore, the sample containing a low bacterial population will produce less number of colonies and vice versa. We must select the isolated colonies for the staining and microscopic examination to study the isolated bacteria’s characteristics.

Conclusion

Therefore, we can conclude that the bacterial isolation is an important method to study and classify the bacteria based on the macroscopic properties (like growth pattern), staining properties, microscopic properties (like colour, shape and size) and biochemical tests. By knowing these characteristics of bacteria, the diagnosis of clinical specimens and the identification of bacteria that are naturally found in the environment becomes easy.


Biochemical reactions:

Gram negative enteric bacilli play an important role in the contamination of food. Hence they are the main causative agents of intestinal infection. Gram negative family includes Shigella, Salmonella, Proteus, Klebsiella,Escherichia,Enterobacter etc. Usually four tests are used for differentiation of the various members of Enterobactericeae. They are Indole test,Methyl red test, Voges proskauer test and Citrate test collectively known as IMViC series of reactions.


Main diseases caused by Staphylococcus aureus

  1. Skin Infections & Surgical wound infections
  2. Osteomyelitis
  3. Food poisoning/gastroenteritis
  4. Toxic shock syndrome
  5. Pneumonia (mainly hospital acquired)
  6. Acute endocarditis
  7. Infective arthritis
  8. Necrotizing fasciitis
  9. Sepsis and Staphylococcal scalded skin syndrome (SSSS)

Chapter 7: Identification and Characterization of Neisseria meningitidis

N. meningitidis are gram-negative, coffee-bean shaped diplococci that may occur intracellularly or extracellularly in PMN leukocytes. N. meningitidis is a fastidious organism, which grows best at 35-37°C with

5% CO2 (or in a candle-jar). It can grow on both a blood agar plate (BAP) and a chocolate agar plate (CAP). Colonies of N. meningitidis are grey and unpigmented on a BAP and appear round, smooth, moist, glistening, and convex, with a clearly defined edge. N. meningitidis appear as large, colorless-to-grey, opaque colonies on a CAP. Prior to identification and characterization testing procedures, isolates should always be inspected for purity of growth and a single colony should be re-streaked, when necessary, to obtain a pure culture. For the following identification and characterization procedures, testing should be performed on 18-24 hour growth from a BAP (Figure 1) or a CAP (Figure 2) at 35-37°C with

The following tests are recommended to confirm the identity of cultures that morphologically appear to be N. meningitidis (Figure 3). N. meningitidis can be identified using Kovac&rsquos oxidase test and carbohydrate utilization. If the oxidase test is positive, carbohydrate utilization testing should be performed. If the carbohydrate utilization test indicates that the isolate may be N. meningitidis, serological tests to identify the serogroup should be performed. This sequence of testing is an efficient way to save costly antisera and time. Additional methods for identification and characterization of N. meningitidis using molecular tools are described in Chapter 10: PCR Methods and Chapter 12: Molecular Methods.

Biosafety Level 2 (BSL-2) practices are required for work involving isolates of N. meningitidis, as this organism presents a potential hazard to laboratory personnel and the surrounding working environment. Please refer to Chapter 4: Biosafety in order to follow the guidelines that have been established for laboratorians working in BSL-2 facilities as many of the tests described in this chapter require opening plates with live cultures and are often performed outside of a biosafety cabinet (BSC). molecular tools are described in Chapter 10: PCR Methods and Chapter 12: Molecular Methods.

Figure 1. N. meningitidis colonies on a BAP

Figure 2. N. meningitidis colonies on a CAP

Figure 3. Flow chart for identification and characterization of a N. meningitidis isolate

  1. Kovac&rsquos oxidase test Kovac&rsquos oxidase test determines the presence of cytochrome oxidase. Kovac&rsquos oxidase reagent, tetramethyl-p-phenylenediamine dihydrochloride, is turned into a purple compound by organisms containing cytochrome c as part of their respiratory chain. This test aids in the recognition of N. meningitidis, but other members of the genus Neisseria, as well as unrelated bacterial species, may also give a positive reaction. Positive and negative quality control (QC) strains should be tested along with the unknown isolates to ensure that the oxidase reagent is working properly.
    1. Preparation of 1% oxidase reagent from oxidase powder To prevent deterioration of stock oxidase powder, the powder should be stored in a tightly sealed desiccator and kept in a cool, dark area. Kovac&rsquos oxidase reagent is intended only for in vitro diagnostic use. Avoid contact with the eyes and skin as it can cause irritation. In case of accidental contact, immediately flush eyes or skin with water for at least 15 minutes.
      1. Prepare a 1.0% Kovac&rsquos oxidase reagent by dissolving 0.1 g of tetramethyl-p-phenylenediamine dihydrochloride into 10 ml of sterile distilled water.
      2. Mix well and then let stand for 15 minutes.
        • The solution should be made fresh daily and the unused portion should be discarded.
        • Alternatively, the reagent could be dispensed into 1 ml aliquots and stored frozen at -20°C. The aliquots should be removed from the freezer and thawed before use. Discard the unused portion each day the reagent is thawed.
      Filter paper method

      Figure 4. Kovac's oxidase test: a negative and positive reaction on filter paper Plate method

      Plate method
      • Do not flood the entire plate as the bacteria exposed to the reagent are usually not viable for subculture.
      • Positive reactions will develop within 10 seconds in the form of a purple color where the bacteria were applied to the treated filter paper. Delayed reactions are unlikely with N. meningitidis.
      • Negative reactions will not produce a color change on the treated filter paper.

      Figure 5. CTA sugar reactions for N. meningitidis with utilization of glucose (dextrose) and maltose, indicated by acid production (color change to yellow), and no utilization of lactose or sucrose

      Table 1. Carbohydrate utilization by some Neisseria and Moraxella spp.

      Acid Production from:
      Table 1. Carbohydrate utilization by some Neisseria and Moraxella spp.
      Organism Glucose 1 Maltose Lactose Sucrose
      Neisseria meningitidis + + &ndash &ndash
      Neisseria lactamica + + + &ndash
      Neisseria gonorrhoeae + 2 &ndash &ndash &ndash
      Neisseria sicca + + &ndash +
      Moraxella catarrhalis &ndash &ndash &ndash &ndash

      Footnotes

      • 1 Glucose may also be referred to as dextrose.
      • 2 Some strains of N. gonorrhoeae are weak acid producers and may appear to be glucosenegative in the CTA medium.
      • Each isolate will require as many sections on the slide as the individual serogroup-specific antisera that will be tested as well as a saline negative control.
      • The instructions specify using a micropipettor with sterilized filtered tips to measure the 10 µl of the 5% formalinized saline to suspend the bacteria. The micropipettor will transfer precise and equal measurements for a proper SASG reaction.
      • If a micropipettor and tips are not available, sterile, disposable 10 µl inoculation loops can be used to transfer 10 µl of the 5% formalinized saline, but often do not deliver accurate amounts (between 5-10 µl).
      • If the bacteria are difficult to suspend directly on the slide, make a moderately milky suspension (comparable to McFarland 6 standard) of the test culture in a small vial with 250 µl of 5% formalinized saline and briefly vortex the suspension to mix and break up any pellets. Add 10 µl of this suspension to the lower portion of the slide.
      • DO NOT use the dropper provided with the antisera because it usually delivers larger amounts than is necessary and can easily be contaminated.
      • If a micropipettor and tips are not available, sterile, disposable 10 µl inoculation loops can be used to transfer 10 µl of the antisera, but often do not deliver accurate amounts (between 5-10 µl).
      • Dispose of the tip or loop used to transfer the antisera to the slide in a waste container after each use to avoid contamination of the antisera. If the source of antisera is contaminated, a new vial must be used.

      Figure 6. Rating the intensity of the agglutination reaction

      • A positive result is designated by a 3+ or 4+ (strong agglutination) within 1-2 minutes, except for serogroup B, which is considered positive with a rating of 2+ or greater.
      • A negative result is designated by a 0 (saline), +/-, 1+ or 2+ (weak agglutination).
      • The serogroup is determined when a positive result occurs with only one of the antisera and not with the saline.
      • If a serogroup is not determined, the isolate is considered NG. The following result combinations are all reported as NG:
        • Agglutination in the saline, regardless of strong reactions with other antisera, characterizes the culture as autoagglutinating.
        • Agglutination with more than one serogroup-specific antisera in the absence of agglutination in saline characterizes the culture as polyagglutinating or cross-reactive.
        • No agglutination with any of the antisera or the saline characterizes the strain as non-reactive.
        1. Repeat the test directly on the slide using growth from another section of the same plate.
        2. Make a cell suspension in a small tube and vortex if the result from SASG directly on the slide is unclear and repeat the test.
        3. Add 20 µl of antisera directly to slide and then add a loop full of organism without diluting the specimen with 5% formalinized saline.
        4. Subculture and retest fresh growth the following day.
        5. If the original plate contains different size colonies, make a subculture for each type of colony and test both cultures the next day. The larger colonies usually indicate better capsule production and therefore better reactivity. However, the smaller colonies will occasionally give a better result.
          • If discrepancies are not immediately resolved, any subsequent SASG repeats should be used in conjunction with control strains.
        • Performed for each new lot of antisera received in the laboratory.
        • Performed biannually after initial QC testing.
        • Repeated if a vial has been exposed to temperatures above 4°C or if there is reason to suspect that the vial has been contaminated since the initial QC was performed.

        Follow the SASG testing procedure to QC each lot of antisera using all reference strains available in the laboratory. Record the results provided on the example QC sheet in Figure 7.

        Reading the QC test results
        • The antiserum must give 3+ or 4+ agglutination with homologous antigens within 1-2 minutes.
        • The antiserum must not react with heterologous N. meningitidis serogroups, with the NG reference strain, or in saline.
        • The antiserum agglutinates with one or more reference strains and/or with the NG reference strain and/or in saline.

        Figure 7. Example QC sheet for testing antisera against all N. meningitidis serogroups


        Medical Microbiology Illustrated

        Medical Microbiology Illustrated presents a detailed description of epidemiology, and the biology of micro-organisms. It discusses the pathogenicity and virulence of microbial agents. It addresses the intrinsic susceptibility or immunity to antimicrobial agents. Some of the topics covered in the book are the types of gram-positive cocci diverse group of aerobic gram-positive bacilli classification and clinical importance of erysipelothrix rhusiopathiae pathogenesis of mycobacterial infection classification of parasitic infections which manifest with fever collection of blood for culture and control of substances hazardous to health. The classification and clinical importance of neisseriaceae is fully covered. The definition and pathogenicity of haemophilus are discussed in detail. The text describes in depth the classification and clinical importance of spiral bacteria. The isolation and identification of fungi are completely presented. A chapter is devoted to the laboratory and serological diagnosis of systemic fungal infections. The book can provide useful information to microbiologists, physicians, laboratory scientists, students, and researchers.

        Medical Microbiology Illustrated presents a detailed description of epidemiology, and the biology of micro-organisms. It discusses the pathogenicity and virulence of microbial agents. It addresses the intrinsic susceptibility or immunity to antimicrobial agents. Some of the topics covered in the book are the types of gram-positive cocci diverse group of aerobic gram-positive bacilli classification and clinical importance of erysipelothrix rhusiopathiae pathogenesis of mycobacterial infection classification of parasitic infections which manifest with fever collection of blood for culture and control of substances hazardous to health. The classification and clinical importance of neisseriaceae is fully covered. The definition and pathogenicity of haemophilus are discussed in detail. The text describes in depth the classification and clinical importance of spiral bacteria. The isolation and identification of fungi are completely presented. A chapter is devoted to the laboratory and serological diagnosis of systemic fungal infections. The book can provide useful information to microbiologists, physicians, laboratory scientists, students, and researchers.


        Identification of Other Streptococcus Species: Streptococcus General Methods

        The blood agar plates used in the manner described above are for checking for purity of cultures. If any of the results are unusual or not expected or the culture is contaminated the test must be repeated.

        Vancomycin resistant streptococci or other unknowns other than leuconostocs and pediococci, which are intrinsically vancomycin resistant.

        AccuProbe-Enterococcus Test

        1. Principle
          The AccuProbe-Enterococcus test is used to aid in the identification of atypical enterococci and to help differentiate between Enterococcus and Lactococcus strains.
        2. Inoculum
          An overnight culture grown on blood agar incubated 35°C in CO2.
        3. Reagents and Materials
          Genprobe Accuprobe Enterococcus Culture Identification Test, GEN-PROBE Inc. San Diego, CA
        4. Procedure
          The test is performed according to the package insert instructions.
        5. Reading and Interpretation
          Automated
        6. Limitations
          Use care with the amount of colonies used. Too many colonies will result in a false positive test.
        7. Quality Control
          Quality controls, positive and negative reactions are determined each day the test is determined. E. faecalis SS1273 and S. sanguinis SS910 are used as the positive and negative controls respectively.

        AccuProbe-Pneumococcus Test

        1. Principle
          The AccuProbe-Pneumococcus test is used to aid in the identification of atypical pneumococci and to help differentiate between viridans Streptococcus strains.
        2. Inoculum
          An overnight culture grown on blood agar incubated 35°C in CO2.
        3. Reagents and Materials
          Genprobe Accuprobe Pneumococcus Culture Identification Test, GEN-PROBE Inc. San Diego, CA
        4. Procedure
          The test is performed according to the package insert instructions.
        5. Reading and Interpretation
          Automated
        6. Limitations
          Use care with the amount of colonies used. Too many colonies will result in a false positive test.
        7. Quality Control
          Quality controls, positive and negative reactions are determined each day the test is determined. S. pneumonia and S. sanguinis SS910 are used as the positive and negative controls respectively.

        Acid Formation in Carbohydrate Broths

        1. Principle
          The ability of bacteria to form acid in some carbohydrate broths and not in others can be used in identification schemes. If the bacteria acidify the carbohydrate, the pH will change and the indicator (brom cresol purple) will turn yellow.
        2. Inoculum
          An overnight culture in Todd Hewitt broth incubated over night at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum.
        3. Reagents and Materials
          1. Heart Infusion broth with 1% carbohydrate and 0.16 brom cresol purple indicator. Most of the carbohydrate broths are commercially available (Remel). An asterisk indicates those that are made by CDC media lab.
          2. Pipet
          1. Inoculate carbohydrate broth tube with 1-3 drops of inoculum. 2. The broth tube is then incubated at 35&EpsilonC for up to 7 days in ambient air. Fastidious organisms may be held up to 14d.

          Arginine Hydrolysis

          1. Principle
            Certain bacteria contain the enzymes to hydrolyze arginine. This hydrolysis results in an alkaline change in the media results in a color change in the media. This test can be used for differentiated different bacteria.
          2. Inoculum
            A drop of Todd Hewitt broth culture grown overnight is the preferred inoculum. Alternatively a suspension in Todd Hewitt broth from growth on a plate or a tiny amount of growth from a plate may be used as the inoculum.
          3. Reagents and Materials
            1. Moeller&rsquos decarboxylase broth containing arginine. The medium is commercially available.
            2. Pipet or loop
            1. Add 1-3 drops of culture suspension to the tube of Moeller&rsquos decarboxylase medium containing arginine
            2. Immediately overlay with sterile mineral oil (about 1 to 2 ml).
            3. The medium is incubated at 35&Epsilon C for up to 7 days in ambient air. (Some fastidious organism may be held up to 14d.)

            Bacitracin Test

            1. Principle
              The bacitracin disk is sensitivity test used to differentiate the beta- hemolytic Streptococcus.
            2. Inoculum
              An overnight culture grown on 5% sheep blood agar incubated 35°C in CO2.
            3. Reagents and Materials
              1. bacitracin &ldquoA&rdquo disk (BBL)
              1. Select a beta-hemolytic colony and heavily inoculate a quadrant of a 5% sheep blood agar plate.
              2. Drop an &ldquoA&rdquo disk in the heaviest zone of inoculation.
              3. Tap disk lightly to ensure that it adheres to the agar.
              4. Incubate plate overnight in CO2 at 35°C.

              Bile Esculin Test

              1. Principle
                A selective and differential medium used in the identification of catalase-negative bacteria. The selective agent bile, inhibits most gram positive bacteria. The enterococci and Streptococcusbovis will grow. Esculin in the medium is hydrolyzed to esculetin and dextrose. The esculetin reacts with ferric chloride in the media to form a black-brown color.
              2. Inoculum
                An overnight culture in Todd Hewitt broth incubated over night at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum. An inoculating loopful of culture may also be used.
              3. Reagents and Materials
                1. Bile esculin slant (Remel)
                1. Inoculate tube with 1 drop of inoculum allowing drop to run down slant. Alternatively, the slant may be inoculated with a loopful of growth from a blood agar plate.
                2. The slant is then incubated at 35&EpsilonC for 2 days in ambient air. Fastidious organisms may be held up to 14d.

                Bile Solubility Test

                1. Principle
                  The purpose of the bile solubility test is to aid in the differentiation of S. pneumoniae from all other alpha-hemolytic streptococci. Sodium deoxycholate (2%) acts on the cell wall of pneumococci resulting in lysis.
                2. Inoculum
                  An overnight culture grown on blood agar incubated 35°C in CO2.
                3. Reagents and Materials
                  1. 2% deoxycholate (CDC Central Services Laboratory, formula #5333)
                  2. physiologic saline pH 7.0 3.13 X 100mm glass tube
                  1. Make a 1.0 ml saline suspension of cells from growth on an agar plate. A turbidity equal to that of 1.0 to 2.0 McFarland density standard should be used.
                  2. After a satisfactory density is achieved, divide the suspension into 2 tubes with approximately 0.5 ml in each.
                  3. Add 0.5 ml of 2% sodium deoxycholate (bile salts) to one tube and 0.5 ml saline to the other tube. Mix by vigorous shaking.
                  4. Incubate the tubes at 35-37&Epsilon C for up to 2 h.

                  Camp Test

                  1. Principle
                    Some bacteria produce CAMP factor (a diffusible extracelluar protein) that synergistically acts with the beta-lysin of Staphylococcus aureus and enhances the lysis of red blood cells. The purpose of the CAMP test is to aid in the identification of nonhemolytic group B streptococci and other ß-hemolytic streptococci.
                  2. Inoculum
                    Growth from a blood agar plate or any solid media.
                  3. Reagents and Materials
                    TSA-sheep blood agar
                  4. Procedure
                    1. The CAMP test is performed on TSA-sheep blood agar. A single streak of ß-lysin producing S. aureus made across the center of the plate. Strain SS-695 (Strep. Lab number is a ß-lysin producing strain of S. aureus.
                    2. A single colony of the unknown strain (beta hemolytic streptococci) is picked up with an inoculating loop and used to make a single streak perpendicular but not touching the S. aureus streak. A 2-3 mm space should remain between the streaks.
                    3. Incubate the inoculated plate normal atmosphere overnight at 35&fnof£C. Group B streptococci and a few other beta-streptococci produce an enhancement of the ß-lysin activity of the S. aureus strain.

                    Catalase Test

                    1. Principle
                      Hydrogen peroxide is used (H2O2) to determine if bacteria produce the enzyme catalase.
                    2. Inoculum
                      Cultures that are grown on a blood free media or a colony grown on a blood agar plate that is carefully transferred to a slide without carry-over of any of the erythrocytes. Cultures are typically grown overnight at 35°C in CO2.
                    3. Reagents and Materials
                      1. Three percent hydrogen peroxide is obtained from a commercial drug store.
                      2. Pipet
                      3. Slides
                      1. The catalase test is best performed by flooding the growth of the bacteria (usually on an agar slant but blood free agar plates can be used) in question with 1.0 ml of 3% hydrogen peroxide and observing for effervescence (bubbling) which indicates a positive test. The bacteria must be grown on blood free medium.
                      2. Modifications of the catalase test may be performed by very carefully removing a colony of growth from a blood agar plate with a plastic needle or wooden applicator stick and transferring the colony to a glass slide. A drop of 3% hydrogen peroxide is added to the colony on the slide and observed for effervescence.

                      Clindamycin Test

                      1. Principle
                        Resistance of bacteria to clindamycin is determined by using a clindamycin disk at a concentration of 2µg/ml. This resistance is useful in differentiating the Lactococcus species.
                      2. Inoculum
                        Growth from a blood agar plate or any solid media.
                      3. Reagents and Materials
                        1. clindamycin disk 2µg/ml
                        2. blood agar plate

                        Esculin Hydrolysis

                        1. Principle
                          A differential medium used in the identification of catalase-negative bacteria. Esculin in the medium is hydrolyzed to esculetin and dextrose. The esculetin reacts with ferric chloride in the media to form a black-brown color.
                        2. Inoculum
                          An overnight culture in Todd Hewitt broth incubated over night at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum. An inoculating loopful of culture from a blood agar plate may also be used.
                        3. Reagents and Materials
                          1. Esculin slant (Remel)
                          1. Inoculate slant tube with 1-3 drops of inoculum allowing drop to run down slant. Alternatively, the slant may be inoculated with a loopful of growth from a blood agar plate.
                          2. The slant is then incubated at 35&EpsilonC for 7 days in ambient air. Fastidious organisms may be held up to 14d.

                          Gas from MRS broth

                          1. Principle
                            The production of gas from glucose is tested in Lactobacillus MRS broth.
                          2. Inoculum
                            An overnight culture in Todd Hewitt broth incubated overnight at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum. An inoculating loopful of culture from a blood agar plate may also be used.
                          3. Reagents and Materials
                            The MRS broth is prepared in the CDC Central Services Laboratory, formula No. 9208. The petroleum jelly is also prepared in the CDC Central Services Laboratory, formula No. 9356.
                          4. Procedure
                            The broth is inoculated with 2 or more colonies from a plate or with 1 to 2 drops of broth culture. The broth is then sealed with melted petroleum jelly and, the tube is incubated at ambient air 37° C up to 7 days.
                          5. Reading and Interpretation
                            Gas production is indicated by the gas formation between the broth and the petroleum jelly plug which pushes the wax plug toward the top of the tube. Small bubbles that may accumulate over the incubation period are not read as positive, only when the wax plug is separated from the broth is the test read positive. Most leuconostoc strains are positive at 24 h but some strains may take longer.
                          6. Limitations
                          7. Quality Control
                            Each new lot of MRS broth prepared by the CDC Central Services Laboratory is tested for positive (gas production) and negative (no gas production) reactions. Leuconostoc mesenteroides strain SS-1238 (ATCC-8293) is used for positive and Streptococcus sanguinis strain SS-910 (ATCC-10556) is used for negative gas production. Results are recorded in the QC log.

                          Gram Stain

                          1. Principle
                            The gram stain is used to differentiate between gram-positive and gram-negative bacteria. Cellular morphology can also be determined. Gram-positive and gram-negative bacteria are both stained by crystal violet. The addition of iodine forms a complex within the cell wall. Addition of a decolorizer removes the stain from gram-negative organisms due to their increased lipid content. These cells are stained pink with the counter stain safranin.
                          2. Inoculum
                            The gram stain can be performed on the growth of any strain grown on any type of media. However, for this group of bacteria (gram-positive cocci), it is best performed on the growth of bacteria in thioglycolate broth at 24h incubation. The staining procedure is modified when preparing the smear from thioglycolate broth. The smear can not be fixed to the slide with hear but must be fixed with methanol.
                          3. Reagents and Materials
                            1. Crystal Violet Stain
                            2. Gram Iodine (Combine Gram Iodine Concentrate to Gram Iodine Diluent)
                            3. Decolorizer Solution
                            4. Methanol
                            5. Slides
                            6. Inoculating loop
                            7. Microscope with Immersion Objective
                            1. Spread single loop of culture from the thioglycolate broth to a microscope slide. Spread the culture over 1/3 to 1/2 to the total area of the slide.
                            2. Allow the smear to air dry. This may take up to 1 hour depending on the temperature and humidity of the room.
                            3. Cover the entire bacterial smear with 3 or 4 drops of methanol to fix the smear and allow to air dry. Again this may take up to an hour.
                            4. Cover the bacterial smear with crystal violet stain and allow to stand 1 minute. Gently was the stain off with cool tap water and drain water from slide.
                            5. Cover the smear with grams iodine and allow to stand 1 minute. Gently wash the iodine off with water and drain the water from the slide.
                            6. Rinse the bacterial smear with decolorizer solution for 10 seconds decolorization is complete when the solution runs clear from the slide. Gently rinse with water and drain the slide.
                            7. Cover the bacterial smear with safranin stain, and allow to stand for 1 minute, then gently wash the stain from the slide.
                            8. Blot the slide dry with absorbent paper and examine the slide under oil immersion lens.

                            Growth at 10C and 45C

                            1. Principle
                              Growth at 10C and 45C is determined in heart infusion broth base medium and can be used as differential test for catalase-negative gram positive cocci.
                            2. Inoculum
                              An overnight culture in Todd Hewitt broth incubated overnight at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum. An inoculating loopful of culture may also be used
                            3. Reagents and Materials
                              1. Heart infusion broth base medium Remel number 061030.
                              1. Two of the broth tubes are inoculated with one or two colonies or one to two drops of an overnight Todd Hewitt broth culture.
                              2. Incubate each tube at the respective temperatures, 10C and 45C. For the 10C incubator, a refrigerator that can be adjusted to hold a temperature of 10C is satisfactory. The refrigerator can be used for storage of media and other materials. For the 45C incubator, a hot water bath that is adjusted to hold a temperature at 45C is best.
                              3. The tests are held a minimum of 7 days and up to 14 days in the case of slow growing strains.

                              Hemolysis

                              1. Principle
                                The hemolytic reaction is particularly useful in the differentiation of the Streptococci. The hemolytic reaction is determined on agar media containing 5% animal blood. The most commonly used base medium is trypticase soy agar and the most commonly used blood is sheep blood. The reason for the use of trypticase soy base is that it supports the growth of all the bacteria listed in Table 2. Other base media may be substituted if control strains of all genera are tested for growth. Sheep blood is used because of the convenience in testing throat swabs for ß-hemolytic streptococci. Sheep blood does not support the growth of Haemophilus haemolyticus which appears similar to streptococci on agar containing rabbit, horse, or human blood.
                              2. Inoculum
                                Pure culture on solid media.
                              3. Reagents and Materials
                                1. Trypticase soy agar plates containing 5% sheep blood are obtained from Becton-Dickinson Microbiology Systems, Cockysville Md., product No. 21261.
                                1. Streak culture for isolation on TSA plate with 5% sheep blood.
                                2. Incubate plate at 35°C in CO2 for 24 h.

                                Hipppurate Hydrolysis Test

                                1. Principle
                                  Some bacteria produce the enzyme hippurate hydrolase which hydrolyzes sodium hippurate to form benzoic acid and glycine. The addition of ferric chloride to benzoic acid forms an insoluble brown ferric benzoate precipitate.
                                2. Inoculum
                                  An overnight culture in Todd Hewitt broth incubated at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum. An inoculating loopful of culture from a blood agar plate may also be used.
                                3. Reagents and Materials
                                  1. Hippurate broth commercial suppliers.
                                  2. Ferric choride (FeCl3) commercial supplies. Labeled as TDA if purchased for bioMereiux
                                  1. The hippurate broth is inoculated with one drop of a fresh (16-20 h) Todd-Hewitt broth culture.
                                  2. The broth is incubated for up to 7 days or until turbid growth is seen at 35&EpsilonC.
                                  3. The tube of broth is then centrifuged to sediment the bacteria.
                                  4. Pipette 0.8 ml the clear supernatant to a small clear tube (13 x 100).
                                  5. Add 0.2 ml of ferric chloride reagent to the supernatant. Mix well.

                                  Lancefield Group Antigen

                                  1. Principle
                                    The purpose of determining the group antigen of ß-hemolytic streptococci is identify the species or species/group of streptococci as originally described by Rebecca Lancefield. Acid extraction is used to remove the serogroup from the cell.
                                  2. Inoculum
                                    An overnight 50 ml culture in Todd Hewitt broth incubated at 35° C.
                                  3. Reagents and Materials
                                    1. 50 ml culture
                                    2. 50 µl capillary tube
                                    3. m-cresol purple
                                    4. 13 X 100 mm test tube
                                    5. boiling water bath
                                    6. centrifuge
                                    7. 1 cc vial
                                    8. Serogrouping Reagents
                                    1. Centrifuge cells in 20 minutes at 2000 rpm to achieve a cell pellet.
                                    2. Aspirate off the supernatant.
                                    3. Add 2-3 drops m-cresol purple. Add .1 N HCl dropwise until a color change to pink. Vortex.
                                    4. Place tube in a boiling water bath for 10 minutes.
                                    5. After allowing to cool, transfer contents to a 13 X100 test tube.
                                    6. Centrifuge at 2800 rpm for 10 minutes to remove any precipitate.
                                    7. Transfer to a clean 13 X 100 mm test tube
                                    8. Add .5 N NaOH dropwise until a change to purple.
                                    9. Centrifuge at 2800 rpm for 10 minutes to remove any precipitate.
                                    10. Transfer to a 1ml screwcap storage vial.
                                    11. Set of capillary tubes by adding approximately 1 cm of serogrouping reagent and 1cm of extract. Careful not to get any space or bubbles between the two!
                                    12. Cap the end with clay and set in clay rack.
                                    13. Observe for up to 30 minutes.

                                    Leucine amino peptidase (LAP)

                                    1. Principle
                                      Some bacteria produce leucine aminopeptidase which hydrolyzes the substrate leucine-&beta-naphthylamide to form &beta-naphthylamine. A pink to red color forms when p-dimethylaminocinnamaldehyde (PYR reagent) is added to &beta-naphthylamine.
                                    2. Inoculum
                                      Strains are grown on blood agar plates overnight at 35°C in CO2 for most gram positive bacteria. More than 1 day of incubation may be necessary for more fastidious genera such as the gemellae, alloiococci, and helcococci. The strains to be tested are grown on a blood agar plate until sufficient growth is seen to heavily inoculate the disks.
                                    3. Reagents and Materials
                                      LAP disk (Remel)
                                      PYR reagent
                                      Loops
                                      Deionized Sterile water
                                    4. Procedure
                                      The procedure that is used in the Streptococcus laboratory is modified from the package insert. The PYR test is usually done simultaneously.
                                      1. Place the disks on blood agar plate in an area of little or no growth or on a slide. The moisture from the plate is usually sufficient to rehydrate the disk. If the disk is placed on a slide, then a tiny drop of sterile deionized water is added. (DO NOT OVERSATURATE THE DISK). It is convenient to place the LAP disk on the left (L=LAP).
                                      2. Using a loop or wooden stick, inoculate the disks heavily. Using two or more loop-fulls of culture is necessary for satisfactory results.
                                      3. Leave the plates with the disks on the bench at room temperature for 10 minutes.
                                      4. Add the detection reagent and read after 3 minutes.

                                      Litmus Milk Test

                                      1. Principle
                                        The purpose of the litmus milk test is to determine the acidification and clot of the milk in this test. This tests aids in the differentiation of the Leuconostoc species.
                                      2. Inoculum
                                        An overnight culture in Todd Hewitt broth incubated at 35° C or a fresh bacterial suspension in Todd Hewitt broth may be used as the inoculum. An inoculating loopful of culture may also be used.
                                      3. Reagents and Materials
                                        Litmus milk is obtained from commercial suppliers using their quality control.
                                      4. Procedure
                                        1. Tubes containing litmus milk are inoculated with one drop of an overnight Todd-Hewitt broth culture.
                                        2. Incubate at 35&EpsilonC for up to 7 days in ambient air. Some fastidious strains may be held for 14 d.

                                        Motility Test

                                        1. Principle
                                          The ability of bacteria to move through a semisolid media is useful in differentiating bacteria. This test is particularly useful in differentiating the enterococci.
                                        2. Inoculum
                                          Strains are grown on blood agar plates overnight at 35°C in CO2 for most gram positive bacteria. More than 1 day of incubation may be necessary for more fastidious genera such as the gemellae, alloiococci, and helcococci.
                                        3. Reagents and Materials
                                          1. motility test medium (Remel, number 061408)
                                          2. inoculating needle
                                          1. The medium is inoculated with an inoculating needle, not a loop. Apply a colony to the end of the needle from the agar plate.
                                          2. The needle is inserted into the center of the medium in the tube for about one inch.
                                          3. The inoculated tube is incubated at 30C in ambient air and incubated until good growth is observed, in most cases 24 to 48 h is sufficient.

                                          6.5% NaCl Tolerance Test

                                          1. Principle
                                            Tolerance tests can be used in the differentiation of microorganisms. Some bacteria can grow in 6.5% NaCl and others are inhibited by these concentrations. Growth in broth containing 6.5% NaCl is determined in heart infusion broth base with the addition of 6% more NaCl. Heart infusion base contains 0.5% NaCl. To make the test easier to read we add 0.5% dextrose and brom cresol purple indicator.
                                          2. Inoculum
                                            A fresh inoculum grow in Todd Hewitt broth is preferred. Alternatively, a small loopful of growth from a blood agar plate may be used.
                                          3. Reagents and Materials
                                            6.5% NaCl broth 5 ml Todd Hewitt broth The 6.5% NaCl broth is prepared by the CDC Central Services Laboratory, formula No. 1707. This formulation is identical to the modified 6.5% NaCl broth described in: Facklam, R. 1973. Comparison of several laboratory media for presumptive identification of enterococci and group D streptococci. Appl. Microbiol. 26:138-145.
                                          4. Procedure
                                            1. One or two colonies or one or two drops of an overnight broth culture is inoculated into the broth containing 6.5% NaCl.
                                            2. The inoculated broth is incubated at 37&EpsilonC in ambient air for up to a week or more depending upon the growth characteristics of the strain being tested. If 2 or 3 days were required for sufficient inoculum then the NaCl tolerance test should be incubated 10 to 14 days. In some cases (most enterococci) the test is positive after overnight incubation.

                                            Optochin Test

                                            1. Principle
                                              The purpose of the optochin test is to confirm the identification S. pneumoniae before serotyping and to aid in the differentiation of S. pneumoniae from viridans streptococci during surveillance studies.
                                            2. Inoculum
                                              Isolated alpha-hemolytic colonies suspected of being pneumococci.
                                            3. Reagents and Materials
                                              1. optochin [email protected] disks purchased from Becton Dickinson Microbiology Systems, Cockeysville, Md.
                                              2. blood agar plates
                                              1. Transferred an isolated colony and streak to a quarter of a blood agar plate
                                              2. Place the optochin &ldquoP&rdquo disk in the upper third of the inoculum. Tap the disk to insure that it stays on the media after the plate is inverted.
                                              3. The plate is incubated overnight at 35-37&EpsilonC in a candle extinction jar or carbon dioxide incubator.

                                              Pigmentation Test

                                              1. Principle
                                                Some bacteria produce pigment. The purpose of the pigmentation test is to aid in the identification of E. casseliflavus, E. mundtii,E. pallens, E. gilvus and E. sulfureus. These enterococci produce a yellow pigment that can be detected on several different media.
                                              2. Inoculum
                                                The unknown Enterococcus strain is grown on trypticase-soy 5% sheep blood agar plate for 24 h in a normal atmosphere at 35&EpsilonC.
                                              3. Reagents and Materials
                                                1. cotton swab
                                                1. Use a cotton swab to pick up a 50 mm smear of bacteria.
                                                2. Examine the swab and smear for a bright yellow color.

                                                Pyridoxal Requirement Test (Vitamin B6)

                                                1. Principle
                                                  Nutritionally variant streptococci (Abiotrophia and Granulicatella) are usually very fastidious and will grow only on supplemented media or enriched chocolate agar. These strains require pyridoxal for growth while non-NVS do not. A final concentration in broth of 0.001% of pyridoxal will support the growth of NVS. An alternative to performing the pyridoxal requirement test is the satellite test.
                                                2. Inoculum
                                                  The cultures are typically received on chocolate agar slants.
                                                3. Reagents and Materials
                                                  1. 0.01% pyridoxal*
                                                  2. 2-TSA-sheep blood agar plate

                                                  * It is convenient to keep a 0.01% solution of pyridoxal in the laboratory. This solution is prepared in purified water and filter sterilized. A 10 ml aliquot is dispensed into sterile tubes. This 0.01% solution should be kept frozen at -20&Epsilon C. The aliquot in use may be stored at 4° C.


                                                  Staphylococcus aureus | Microbiology Unknown Lab Report

                                                  It is important to understand why a person would want to identify between different bacteria. In order for an individual to receive treatment for a certain bacterium, this bacterium must first be identified. This is done because some treatments may fight against one bacterium, while another treatment may not work to fight off a different bacterium. The purpose for this study is for the microorganism to be identified. The study below was performed by following the different techniques and methods taught in the microbiology lab class throughout the semester.


                                                  MATERIALS AND METHODS

                                                  After a semester of learning different techniques and methods to identify between different bacteria, the instructor distributed a test tube labeled 103 with an unknown substance inside. The goal is to correctly identify two different bacteria, one being Gram positive and the other Gram negative. In order to identify these bacteria, a series of different laboratory tests must be done. In addition to using sterile technique, the procedures for these tests were followed via the lab manual for general microbiology created by McDonald, Thoele, Salsgiver, and Gero (1), unless otherwise stated in this report.

                                                  The first thing to be done is to try and isolate the two bacteria with the goal of obtaining a pure culture. This is done by streaking a Trypticase Soy Agar (TSA) plate using the streak method as stated in the lab manual. After incubating the TSA plate for 24-48 hours the bacterium grew and their characteristics were recorded. Since there was not a distinct difference between two colonies, the same method was used again on another TSA plate. After checking back in another 24-48 hours, two different distinct colonies grew in the agar plate and from this, another procedure was done. In order to obtain a pure culture, two TSA plates were labeled one with Unknown “A” and the other Unknown “B”. Each colony was then isolated once again using the streak method from the lab manual and then put into the incubator. Once checking and recording the results two days later, the next step was to perform a gram stain on pure culture “A” and pure culture “B”. While practicing sterile technique is important for each procedure to reduce the risk of contamination, it is also extremely important to be extra careful when doing a gram stain that you use the proper technique provided for you in the lab manual. This is because, not only does a gram stain tell whether the bacterium is gram positive or negative, it also tells what shape the bacterium is. It was concluded that unknown “A” was gram positive cocci and unknown “B” was gram negative rods. After this was determined, specific biochemical tests were performed. These biochemical tests were determined by using the Unknown Chart given out by the lab instructor. The following Tables and Flowcharts give a guideline as to which tests were performed and there outcomes for both Gram Positive and Gram Negative bacteria.

                                                  The following tests were performed on the Unknown “A” (Gram Positive)

                                                  The following tests were performed on the Unknown “B” (Gram Negative)

                                                  Unknown 103: Unknown A (Gram Positive)

                                                  After evaluating a pure culture of Unknown “A”, it was determined there was growth that consisted of a medium sized yellow to cream colored colony. After doing a gram stain, it was determined that this bacterium was Gram positive cocci. At this point a urea test was performed. An inoculating loop was sterilized, and the urea test tube was inoculated with the bacteria. Please see Table 1 for a complete list of each test, purpose, reagents, observations and results for each test conducted. Flowchart 1 is also available for the results of this unknown.

                                                  Unknown 103: Unknown B (Gram Negative)

                                                  After evaluating a pure culture of Unknown “B”, it was determined the growth of this bacteria to be a colony of very small dots, white in color. When doing a gram stain the results showed that the bacterium was Gram negative rods. A Mannitol Salt Agar plate was obtained in order to test for mannitol fermentation. An inoculating loop was sterilized, a sample of Unknown “B” was collected and a streak was made on the agar plate. Please see Table 2 for a complete list of each test, purpose, reagents, observations and results for each test performed on Unknown “B”. Flowchart 2 is also available for the results of this unknown.

                                                  TABLE 1. Unknown A: Gram + bacteria

                                                  TEST PURPOSE REAGENTS OBSERVATIONS RESULTS
                                                  Gram Stain Determine the shape of the bacterium/whether it’s gram +/- Crystal Violet, Iodine, Safranin and Alcohol Purple cocci Gram positive cocci
                                                  Urea test Determine if bacteria breaks down urea with urease None No color change. Tube remained yellow Negative for urease
                                                  Nitrate test Determine if bacteria can reduce nitrate to nitrite Reagent A&B, Zinc Test tube turned red after adding Reagent A Positive for nitrate
                                                  Mannitol test Determine if bacteria can ferment mannitol none Agar turned from a red color to a yellow color Positive for mannitol

                                                  TABLE 2. Unknown B: Gram – bacteria

                                                  TEST PURPOSE REAGENTS OBSERVATIONS RESULTS
                                                  Gram Stain Determine the shape of the bacterium/whether it’s gram +/- Crystal Violet, Grams Iodine, Gram Safranin and Alcohol Reddish pink rods Gram negative rods
                                                  Mannitol test Determine if bacteria can ferment mannitol none One side of agar turned yellow, while the other remained red Positive for mannitol
                                                  Methyl Red test Determines if bacteria can produce acid that ferment glucose Methyl Red Color change from yellow to red Positive for Methyl Red
                                                  Indole test See if a bacterium has tryptophanase to convert tryptophan to indole. none Test tube stayed the same color as it was before being inoculated Negative for tryptophanase

                                                  UNKNOWN #103 Unknown “A”(Gram Positive)

                                                  Staphylococcus aureus

                                                  Staphylococcus epidermidis

                                                  Enterococcus faecalis

                                                  Urea Test (negative result)

                                                  Positive Negative

                                                  Staphylococcus epidermidis Staphylococcus aureus

                                                  Enterococcus faecalis

                                                  Nitrate test (positive result)

                                                  Positive Negative

                                                  Staphylococcus aureus Enterococcus faecalis

                                                  Mannitol Test (positive result)

                                                  Staphylococcus aureus

                                                  UNKNOWN 103: Unknown “B” (Gram Negative)

                                                  Mannitol Agar (MSA) (positive result)

                                                  Escherichia coli Proteus vulgaris

                                                  Klebsiella pneumoniae Pseudomonas aeruginosa

                                                  Methyl Red test (positive result)

                                                  Escherichia coli Enterobacter aerogenes

                                                  Klebsiella pneumoniae

                                                  Indole test (negative result)

                                                  Escherichia coli Klebsiella pneumoniae

                                                  DISCUSSION/CONCLUSIONS

                                                  After doing a gram stain on the bacteria, it was determined that it was gram positive cocci. The next step was to go on with a second procedure, which was a Urea test. The object of this test was to find out which bacteria can or cannot produce urease to break down urea. The negative results of this test narrowed the search down to Staphylococcus aureus or Enterococcus faecalis. From there, a Nitrate test was performed. This test shows whether or not a bacterium can reduce nitrate to nitrite. A positive reaction for nitrate occurs when reagents a and b are added and once reagent a is added, the test tube turns red in color. Since both of these tests were positive, this eliminated Enterococcus faecalis from the list. After eliminating this bacterium, the only one left was Staphylococcus aureus. To be sure of the results, a Mannitol test was done to check to see if the bacteria could ferment mannitol. After incubating for 48 hours, the agar plate turned yellow on one side and stayed red on the other. This is an indication of a positive test. Therefore, this ensures that Unknown “A” is indeed Staphylococcus aureus.

                                                  After doing a gram stain on the bacterium, it was determined that this bacterium was a gram negative. The next step was to continue with another test. A Mannitol agar plate was obtained and inoculated with the bacterium. The purpose for this is to see if the bacterium can ferment mannitol. Once observing that half of the plate was yellow, this means the results were positive for mannitol. By doing this test, Proteus vulgaris and Pseudomonas aeruginosa were eliminated from the list. Since Escherichia coli, Klebsiella pneumoniae, and Enterobacter aerogenes are left, another test was performed to eliminate more bacteria. The next test was a Methyl Red test. This is used to find out if a bacterium can produce acid that ferments glucose. After incubation and adding the methyl red drops to the results, the test tube turned red in color a positive test for methyl red. This leaves either Escherichia coli or Klebsiella pneumoniae. The final test done was an Indole test. This test is used to see if a bacterium has the enzyme tryptophanase to convert tryptophan to indole. When this happens, the top of the test tube will turn a reddish pink color. Once checking the results 24-48 hours later, the test tube had not changed since before it was put into the incubator which means the test was negative for Indole. Therefore, this means that Unknown “B” is Klebsiella pneumoniae.


                                                  Staphylococcus aureus (S. aureus) is a gram positive bacterium that when looked at under a microscope it appears to be a cluster of what looks like purple circles. This shape is known as cocci. When grown on a TSA plate, Staphylococcus aureus appears to be yellow to opaque in color. S. aureus is known as one of the most resistant bacterium to multiple antibiotics and considered the most pathogenic. Everyone is susceptible to S. aureus with one way of transmission being from foods such as chicken, eggs, meat, and tuna which can all cause food poisoning. Another way of transmitting the disease would be from your skin. Staphylococcus aureus resides on a person’s skin on a daily basis and when the barrier is broken from things such as cuts or wounds, this then provides an excellent entry way for the bacterium. Drug users are also extremely susceptible to this bacterium because they may choose to inject themselves with needles which can cause the likeliness of the bacterium entering your body (3). It has also been found that Staphylococcus aureus plays a huge role in Methicillin resistant Staphylococcal aureus otherwise termed MRSA. This bacterium can become resistant to many antibiotics such as methicillin, cephalosporins and erythromycin which make it much more difficult to treat. In order to try and treat MRSA vancomycin is administered to the patient. Even with this drug, researchers have found that MRSA is also becoming resistant to vancomycin as well. The prevention of this bacterium could help to minimize nosocomial infections by practicing good hygiene and appropriate cleansing of surgical incisions and burns (2).

                                                  In conclusion, it was found that the gram positive bacterium was indeed Staphylococcus aureus and the gram negative bacterium was Klebsiella pneumoniae. One problem that was difficult to overcome was maintaining sterile technique while inoculating both agar plates and test tubes. The isolation was difficult to do because two attempts were made to obtain a pure culture. After three weeks of performing different tests, the results show the correct identification of the unknown bacteria.


                                                  Watch the video: How to identify u0026 Confirm Staphylococcus Aureus in laboratory??? (January 2022).